Controls for implementing multiplex analysis methods

ABSTRACT

The present invention relates to controls which may be used to secure the results of multiplex analysis methods. The present invention thus relates to solid supports comprising one or several controls and their use in multiplex analysis methods to detect several analytes potentially present in a sample.

TECHNICAL FIELD

The present invention relates to controls able to be used to secure theresults of multiplex analysis methods comprising one or several steps.

BACKGROUND OF THE INVENTION

A multiplex analysis method allows the simultaneous detection of thepotential presence of several analytes within a same sample. Multiplexanalysis has several advantages, such as time savings by making itpossible to analyze several analytes at the same time, lower consumptionof reagents and consumables, as well as a lower quantity sample neededto detect analytes.

It is common to use one or several controls to validate the resultsobtained at the end of the analysis method seeking to detect thepresence of one or several analytes. The reliability of the resultsprovided by an analysis device is in fact a major issue, in particularwhen it involves analyses intended for medical diagnostics and/or thequalification of transfusion donations.

One positive control traditionally used consists of verifying thedetection of a known compound, used in a known quantity and thatcorresponds to an analyte whereof the potential presence is sought in agiven sample. However, this type of control makes it possible tovalidate only the overall implementation of the analysis method and doesnot make it possible to validate each step of the method.

Furthermore, other types of control may be used. For example, documentWO2004/046685 uses controls to verify the quality of the reagents usedin immuno-histochemical tests. To that end, a controlled slidecomprising a series of solutions of different control compounds is used.Positive coloring of the control side at a control compound must only beobserved if a specific antibody of said control compound or a reporteror substrate of said control compound is used during the immuno-markingmethod. This method for verifying the quality of the reagents requiresthe use of a large number of control compounds on the control slide.

Indeed, there is currently no simple means making it possible to controlall of the steps in the case of a multiplex analysis method: inparticular, the deposition of the sample, the deposition of thereagents, the different water and incubation cycles. It is, however,crucial in the field of blood transfusions and medical diagnostics toachieve a high level of security and traceability.

Thus, there is a real need to provide solutions making it possible toguarantee the reliability of the results obtained during theimplementation of multiplex analysis method and that in particular makeit possible to validate each step of the method.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the revelation by the inventors ofcontrol means making it possible to guarantee the reliability of theobtained results, by verifying that each step of a multiplex analysismethod has taken place correctly (and not by only verifying an overallimplementation of the method). Thus, for the first time, the inventorsare providing controls that make it possible to validate the depositionof the sample itself, and more generally the different steps of themethod.

By combining only two or three controls according to the invention, itis thus possible to validate the deposition step of the sample, thedeposition step(s) of the specific detection ligands of the analytes tobe detected in the sample, the different washing and incubation steps,and if applicable, the deposition step of a reporter of a detectionmarker and/or the deposition step of a substrate of the marker coupledto said reporter.

In the multiplex analysis method according to the invention, only two orthree controls are necessary to validate each step of the entiremultiplex method.

Aside from the validation of the results, the use of the controlsaccording to the invention also makes it possible to identify potentialfaults existing during the implementation of the multiplex analysismethod, and for example to modify the corresponding step(s) to improvethe implementation of the analysis method.

The present invention also has the advantage of being easy to implement,requiring only a limited number of controls, not adding additional stepsto the analysis method and not requiring the use of additional equipment(for example, not requiring the use of a spectrophotometer). Indeed, thesteps of the analysis method are done in a single location (for example,the tube with the well in which the sample is placed) and the controls,for example in the form of spots or beads, are processed at the sametime as the spots or beads used to detect analytes.

The first type of control according to the invention is the control ofthe deposition of a sample that makes it possible to verify thedeposition of the sample. In certain embodiments outlined below, thecontrol of the deposition of the sample also makes it possible to verifythe deposition of one or several analyte detection ligands.

“Deposition of the sample or an additive” or “addition of the sample oran additive” refers to the placement of the sample or at least oneadditive in the presence of the compounds of interest fixed on a solidsupport.

“At least one”, within the meaning of the present application, refers toone or several, several in particular meaning two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen or more than sixteen.

“Deposition of a detection ligand, a reporter or a substrate” or“addition of a detection ligand, a reporter or a substrate” refers tothe placement of a detection ligand, a reporter or a substrate in thepresence of compounds of interest fixed on a solid support and anycompounds fixed on said compounds of interest.

The second type of control is the control of the deposition of adetection ligand of an analyte that makes it possible to verify thedeposition of one or more specific detection ligands (for example amixture of detection ligands) of analytes to be detected in the sample.This control also makes it possible to validate the incubation andwashing steps.

The third type of control is the control of the deposition of a reporterthat makes it possible to verify that the developing step(s) have takenplace correctly. The control of the deposition of a reporter is usefulin case of indirect marking of the detection ligands.

Lastly, the controls according to the invention are particularlyappropriate for carrying out multiplex analysis methods in micro-arrays,for example on a solid support of the microplate type, or in liquidchip, for example on a solid support of the bead type.

Sample

The sample to be analyzed is preferably a biological sample.

The biological sample may be a biological fluid, such as a sample ofblood, blood derivatives (such as plasma or serum), urine, cerebrospinalfluid, saliva, or a tissue sample, such as a tissue obtained by biopsy,a cell or set of cells, a plant extract, or combinations thereof.

A blood derivative refers to any product, in particular fluid, obtainedfrom a blood sample.

The sample to be analyzed may also be a culture medium and/or a culturesupernatant.

Before being analyzed, the sample may undergo one or several priortreatment steps, such as dilution, centrifugation, heat and/or chemicaltreatment, cell lysis (for example by one or several chaotropic agents,one or several reducing agents and/or by heating), extraction, PCR(Polymerase Chain Reaction), addition of an unmarked detection ligand orcombinations thereof. The addition of an unmarked detection ligand is inparticular useful to implement a neutralization test, which in itself isa test known by those skilled in the art.

The sample may also be a mixture of at least two samples that may be ofthe same nature or different natures.

Examples of mixtures of samples of different natures are a mixture ofblood and serum, a mixture of blood and plasma, a mixture of serum andplasma, or a mixture of blood, serum and plasma.

One preferred sample according to the invention is a sample or mixtureof samples of blood and/or blood derivatives.

Analyte

An analyte to be detected in a sample may be any type of compound,natural or synthetic, that one wishes to detect and/or quantify in asample.

An analyte may for example be a protein, a peptide, a glycoprotein, acarbohydrate, a lipid, a cell, an organelle, a virus or a nucleic acid.

The cell may be an animal cell, a plant cell, a bacteria cell, aprotozoa, a metazoan cell, a yeast cell, a fungus cell or a protozoa.

A nucleic acid designates a polymer of nucleotides linked byphosphodiester bonds, such as a deoxyribonucleic acid (DNA), aribonucleic acid (RNA) or an analogue thereof, such as phosphorothioatesor thioesters, in single-strand or double-stranded form.

An analyte or at least one of the analytes or the analytes is (are) forexample chosen from the group consisting of an antigen, an antibody, anantibody fragment, a hapten, a hormone, a hormone receptor, an enzyme,or a nucleic acid.

In one preferred embodiment, the analyte(s) are not nucleic acids.

The analyte(s) are for example selected from the group consisting of anantigen, an antibody, an antibody fragment, a hapten, a hormone, ahormone receptor and an enzyme.

Here, “antigen” refers to a natural, recombinant or synthetic moleculerecognized by antibodies or cells of the immune system and capable ofcausing an immune response when it is presented under appropriateconditions to the immune system of a host. This may be a molecule, inparticular a polypeptide, comprising or consisting of at least oneepitope that may be linear or conformational. The term “linear epitope”refers to a polypeptide, in particular a peptide, comprising orgenerally consisting of 3 to 15, more generally 5 to 15 amino acids,preferably at least 6, 8, 10 or 12 amino acids, capable of binding to anantibody molecule against said antigen. The term “conformationalepitope” refers to a three-dimensional structure recognized by anantibody and determined by the juxtaposition of several amino acids inspace, which may be noncontiguous in the peptide sequence of the protein(or polypeptide) against which this antibody is directed, but which, dueto the folding of the polypeptide chain, find themselves dose to oneanother in space, and can thus form a pattern that may be recognized byan antibody.

An antigen is for example a protein (in particular a native orrecombinant protein), a peptide (for example, a synthetic peptide), aglycoprotein, a carbohydrate or a lipid, said peptide being able to beassociated or not associated with a carrier molecule, for example BSA(bovine serum albumin).

In the present application, a “carrier molecule” in particular refers toa protein or carbohydrate carrier molecule. A carrier molecule may be apolypeptide (in particular protein or a peptide), which may or may notbe natural (for example, a recombinant protein or a synthetic peptide),a functionalized polymer (such as dextran, polysaccharide or polylysine)or a mixed copolymer (in particular a copolymer of different aminoacids, for example a lysine-tyrosine copolymer). A carrier molecule maybe an antibody (in particular a monoclonal antibody or a polyclonalantibody), for example an immunoglobulin.

One example carrier molecule is BSA.

In one specific embodiment, the carrier molecule is not an antibody.

“Hapten” here refers to a molecule with a low molecular weight capableof being recognized by the immune system, but which is immunogenic onlywhen it is coupled to a carrier molecule.

An analyte or at least one of the analytes is preferably a compoundmaking it possible to diagnose a condition in a subject, which may ormay not be pathological, or to diagnose the risks of developing acondition, which may or may not be pathological. An example of anon-pathological condition is a pregnancy.

The subject may be a human, a non-human animal or a plant. The non-humananimal is preferably a mammal, such as a cat, dog, monkey, rabbit, mouseor rat.

The term “human” is used broadly and in particular designates a man or awoman of any age, such as an infant, a child, an adolescent, an adult oran elderly person.

When the analyte or at least one of the analytes is an antigen, it ispreferably an antigen making it possible to diagnose an infection, forexample an infection caused by a virus, a bacteria, a fungus or aparasite.

When the analyte or at least one of the analytes is an antibody, it ispreferably an antibody making it possible to diagnose an infection, forexample an infection caused by a virus, a bacteria, a fungus or aparasite.

Typically, this may involve one or several antigens and/or one orseveral antibodies of:

-   -   a virus, such as HIV (Human Immunodeficiency Virus), in        particular HIV-1 or HIV-2, HBV (Hepatitis B Virus), HCV        (Hepatitis C Virus), HPV (Human Papilloma Virus), HTLV (Human        T-Lymphotropic Virus), in particular HTLV-I or HTLV-II,    -   a parasite, such as a parasite that may cause toxoplasmosis (in        particular Toxoplasma gondii), malaria (in particular a parasite        of the Plasmodium genus, for example Plasmodium falciparum,        Plasmodium vivax, Plasmodium ovale, Plasmodium malariae or        Plasmodium knowlesi) or Chagas disease (in particular        Trypanosoma cruzi) in humans or non-human animals, or    -   a bacteria, such as a bacteria able to cause syphilis (Treponema        pallidum) or Lyme disease (in particular a bacteria from the        Borrelia genus) in humans or non-human animals.

“Parasite” here refers to a metazoan or a protozoa acting as parasitewith respect to a body and causing parasitosis. A parasite within themeaning of the invention is therefore not a virus, a bacteria or afungus.

The analyte or at least one of the analytes may also be a marker fordisease, such as a marker of a cardiovascular disease or a diabetesmarker, a marker of the evolution of the disease, such as hepatitis, amarker of the evolution of an infection caused by a virus, a bacteria, afungus or a parasite, a marker of resistance to a treatment, for exampleto an antiviral treatment, an antibiotic treatment or a cancertreatment.

Several (for example, two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, fifteen or more than fifteen)analytes as described in the present application may be detectedsimultaneously in a sample during a multiplex analysis method. This maymake it possible to diagnose, in a same sample, one or severalinfections or diseases, the evolution of an infection or disease, acondition (pathological or not), a risk of developing a condition(pathological or not) or a marker of resistance to a treatment in asubject.

The analytes detected during a multiplex analysis method may be of thesame nature (for example only antibodies or only) or of differentnatures (for example, at least one antigen and at least one antibody).

Preferably, the analyte(s) to be detected are not marked with adetection marker.

In one preferred embodiment, the analyte(s) to be detected are chosenfrom among an antibody and/or an antigen.

Control Compound

Here, “control compound” refers to a compound naturally present in thesample to be analyzed, preferably at a concentration detectable in allof the samples of the same nature.

The compound naturally present in the sample to be analyzed can be acompound initially present in the sample or a derivative of a compoundinitially present in the sample.

A derivative of a compound initially present in the sample can beobtained at the end of one or several treatment steps of the sampleprior to the analysis method. These steps are in particular as definedabove, for example heat and/or chemical treatment, cell lysis,extraction, PCR (Polymerase Chain Reaction), addition of an unmarkeddetection ligand or combinations thereof.

A derivative of a compound initially present in the sample is forexample a PCR product and/or a compound modified by a heat and/orchemical treatment, cell lysis, the addition of an unmarked detectionligand or combinations thereof.

Preferably, a compound naturally present in the sample to be analyzed isa compound initially present in the sample. In this case, if one orseveral treatment steps of the sample prior to the analysis method arecarried out, the compound is therefore present before any priortreatment step of the sample.

A compound naturally present in the sample to be analyzed does notinclude a compound present in the sample at the time of the analysis,but that has been added or that is derived from a compound added to thesample during one or several prior treatment steps of the sample.

When the sample is a mixture of at least two samples, the controlcompound is a compound naturally present in at least one of saidsamples, preferably in each sample of the mixture.

“Sample of the same nature” refers to a sample of the same originwithdrawn in substantially the same way in different subjects or in asame subject at different time intervals and, if applicable, havingundergone the same treatment step(s).

A “detectable concentration” is a concentration making it possible todetect the presence of a control compound during a test, in particularan immunological test of the sandwich type in which a control compoundbonds to a capture antibody fixed on a solid support and is detected bya marked detection antibody.

Preferably, the concentration of a control compound varies little insamples of the same nature.

The expression “the concentration of a control compound varies little insamples of the same nature” here means a concentration that varies byless than 60%, preferably less than 40%, more preferably less than 20%from one sample to another, the concentration being given in μg/mL.

In one advantageous embodiment, a control compound is present in thesample in a concentration of the same order of magnitude as theconcentration of the analyte(s) to be detected, when these analytes arepresent. Preferably, a control compound is present in the sample in aconcentration of the same order of magnitude as the concentration ofeach of the analytes to be detected.

The concentration of a control compound is of the same order ofmagnitude as the concentration of each of an analyte to be measured.

Preferably, the concentration of the control compound is more than 5decades lower or higher than that of said analyte to be assayed, when itis present in the sample, preferably at least 4 decades, more preferablyat least 3 decades.

For example, the concentration of a control compound is no more than 100μg/mL, preferably no more than 10 μg/mL, more preferably no more than 1μg/mL, for a concentration of an analyte to be assayed of 1 ng/mL.

Preferably, the concentration of the control compound is of the sameorder of magnitude as the concentration of an analyte, when thedeviation between the concentration of the analyte in a sample to beanalyzed that contains this analyte and that of the control compound isno more than 5 decades, preferably no more than 4 decades, morepreferably no more than 3 decades.

In the case of a sample of human or animal origin, a control compoundpreferably has a structure retained within the human or animalpopulation in question for the analysis. In one preferred embodiment, acontrol compound has no polymorphism within the considered population.

The control compound preferably is not a marker of a disease.

When at least one analyte is detected by an antibody, a control compoundis preferably a compound detectable by an antibody.

The control compound is preferably not altered by any possible priortreatment step(s) of the sample, so as to be detectable by an antibody.

The control compound may be a molecule, or a complex of at least twomolecules, which may be identical or different.

As an example, when the sample to be analyzed is a blood or plasmasample, in particular human blood or human plasma, a control compoundmay be the soluble a coagulation factor, for example a coagulationfactor chosen from factors VIII, IX, X, XI, XII or XIII.

In one specific embodiment, a control compound is not factor XIII.

In one particular embodiment, a control compound is not animmunoglobulin of type G (IgG), in particular a human IgG, and/or is notan immunoglobulin of type M (IgM), in particular a human IgM, or moregenerally, is not an immunoglobulin, in particular a humanimmunoglobulin.

The soluble receptor of the transferrin complexed to the transferrin isa complex comprising two molecules of the soluble receptor of thetransferrin and two transferrin molecules.

For example, the soluble receptor of the transferrin complexed to thetransferrin is generally present in a concentration comprised from 0.8μg/mL to 4 μg/mL in the samples to be analyzed coming from a humansubject, independently of the condition of the subject.

When the concentration of a control compound may vary, for example basedon the nature of the sample and/or the condition of the subject fromwhich the sample was taken, it may be advantageous to use at least twodifferent control compounds.

Additive

An additive is a compound or a set of compounds that is (are) notpresent in the sample, i.e., that is (are) not initially present in thesample or that is (are) not derived from a compound initially present inthe sample.

An additive may in particular be an antigen or a hapten, said antigen orsaid hapten being able to be coupled or not coupled to a carriermolecule.

As an example, for a human or animal biological sample, a compound orone of the compounds that is not present in the sample to be analyzed isfor example selected from the group consisting of digoxigenin, a planthormone, an alkaloid, a plant steroid, a nucleic acid and a pesticide.

In one preferred embodiment, the additive is not a nucleic acid.

Digoxigenin is a steroid extracted from certain plants.

A pesticide is for example an insecticide, such as an organophosphate oran organochlorine, and herbicide, such as triazine or phenyl-urea, orcombinations thereof.

One preferred additive according to the invention is digoxigenin coupledto a carrier molecule, an auxin coupled to a carrier molecule, atriazine coupled to a carrier molecule, said carrier molecule forexample being BSA.

One preferred additive according to the invention is digoxigenin coupledto BSA.

Furthermore, the additive does not interfere with the detection of theanalytes during the implementation of a multiplex analysis method. Inparticular, the additive does not interfere with the analytes to bedetected, the capture ligand(s) used, the detection ligand(s) used, thecontrol compound(s), if applicable the reporter(s), if applicable thesubstrate(s), and the detection of the signal.

In one particular embodiment, the additive does not comprise or consistof biotin or an analogue of biotin and/or avidin or an analogue ofavidin (in particular streptavidin or neutravidin), said biotin, avidinor one of their analogues being grafted or not grafted on a carriermolecule.

Capture Ligand

A capture ligand is a compound fixed on a solid support, in particularat a spot or the surface of a bead.

A capture ligand is preferably an antibody or antigen that is fixed onthe solid support, in particular at a spot or the surface of a bead.

A capture ligand may be specific to an analyte to be detected in thesample, a control compound or an additive.

A capture ligand may be an antibody, an antigen, a peptide, acarbohydrate, a lipid or a nucleic acid.

In one preferred embodiment, the capture ligand is not a nucleic acid.

In one preferred embodiment, the capture ligand is selected from thegroup consisting of an antibody, an antigen, a peptide, a carbohydrateand a lipid.

A capture ligand is preferably an antibody or antigen.

When a capture ligand is an antibody, it for example involves amonoclonal antibody or a polyclonal antibody.

Detection Ligand

A detection ligand is intended to reveal the presence of a compound towhich it is specific.

A detection ligand may be an antibody, an antigen, a peptide, acarbohydrate, a lipid or a nucleic acid.

In one preferred embodiment, the detection ligand is not a nucleic acid.

In one preferred embodiment, the detection ligand is selected from thegroup consisting of an antibody, an antigen, a peptide, a carbohydrateand a lipid.

A detection ligand is preferably an antibody or an antigen.

When a detection ligand is an antibody, it for example involves amonoclonal antibody or a polyclonal antibody.

A detection ligand is preferably a marked detection ligand, i.e., adetection ligand to which a detection marker (which may for example bebiotin or a peroxidase) is attached, covalently or non-covalently.

When a detection ligand is not marked, its detection may be obtained byusing a specific marked antibody of said detection ligand.

A detection ligand may be specific to an analyte to be detected in thesample, a control compound or an additive.

A detection ligand may be identical to the used capture ligand or one ofthe used capture ligands, with the exception of any presence of adetection marker, and/or bind to the compound to which it is specific atthe same zone as that bonded by the capture ligand or one of the captureligands. In this case, if said capture ligand and said detection ligandare antibodies, it then involves a “homologous sandwich”.

A capture ligand and the detection ligand or one of the detectionligands can be specific to separate zones at the compound to which theyare specific, so as to avoid competition of the capture ligand and thedetection ligand with respect to the compound to which they arespecific, due to a steric hindrance. In this case, if said detectionligand and said capture ligand are antibodies, it then involves a“heterologous sandwich”.

In one preferred embodiment, a detection ligand and a capture ligandspecific to a same compound do not bond to the same location on saidcompound. More preferably, said detection ligand bonds to a zone of saidcompound that is far from the binding zone with said capture ligand.

In another preferred embodiment, a detection ligand is identical to acapture ligand, with the exception of any presence of a detectionmarker, and/or bonds to the compound to which it is specific at the samezone as that bonded by said capture ligand, in particular when thecompound to which it is specific is in the form of a complex having atleast two identical bonding zones.

Detection Marker

A detection marker may be a direct marker or an indirect marker.

A direct marker is a marker whose signal can be detected directly, i.e.,without requiring the prior addition of a reporter.

A direct marker is for example selected from the group consisting of alanthanide, a luminescent compound, a transition metal such asruthenium, a chromogenic, and colored, fluorescent or luminescentnanoparticles.

In the present application, a “luminescent compound” in particulardesignates an electroluminescent, thermoluminescence or (preferably)chemiluminescent compound.

One example luminescent compound (more specifically, thermoluminescentcompound) that may be used as a direct marker consists of silicananoparticles comprising (for example doped with) molecules of adioxetane compound, in particular the 1,2-dioxetane compound, or aderivative of a dioxetane compound, for example a derivative of1,2-dioxetane.

An indirect marker is a marker for which detection of the signalrequires the prior addition of a reporter, and if applicable, theaddition of a substrate of the marker coupled to said reporter.

A reporter is a substrate of the indirect marker or a moleculespecifically bonding to the indirect marker, said molecule itself beinga direct or indirect marker or itself being coupled to a direct orindirect marker.

An indirect marker may for example be an enzyme (in particular, anenzyme producing a luminescent compound from a substrate), biotin,avidin, streptravidin, neutravidin, a hapten, an antigen or an antibody.

A reporter of an enzyme is for example a substrate of said enzyme.

A reporter of a luminescent compound is for example an enzyme or acatalyst.

A reporter of the biotin is, for example, avidin, streptavidin orneutravidin, preferably coupled with a direct marker or an indirectmarker, such as an enzyme or a catalyst.

An example enzyme is peroxidase, for example horseradish peroxidase(HRP).

One preferred biotin reporter according to the invention is streptavidincoupled with a peroxidase, preferably horseradish peroxidase.

In one particular embodiment of the invention, the detection marker orone of the detection markers used is or has as substrate, luminol(3-aminophthalhydrazide, also called5-amino-2,3-dihydro-phthalazine-1,4-dione, molecular formula C₈H₇N₃O₂),isoluminol (also called 4-aminophthalhydrazide), an acridine,coelenterazine, dioxetane or peroxyoxalic compound, or one of theirderivatives, and in particular a compound described in the publicationDodeigne C. et al (2000), Talanta 51, 415-439, “Chemiluminescence asdiagnostic tool. A review”.

According to one preferred embodiment of the invention, the detectionmarker or one of the detection markers used has, as substrate, luminol,isoluminol, or one of their derivatives.

A derivative of luminol or isoluminol is preferably a molecule obtainedfrom the luminol or the isoluminol, respectively, through all possiblemodification(s) (for example, chemical and/or enzymatic). A derivativeof luminol or isoluminol is for example a substrate of a peroxidaseenzyme, the reaction of said peroxidase enzyme with said derivative ofthe luminol or the isoluminol making it possible to produce achemiluminescent compound.

A derivative of the isoluminol may for example be aminoethylisoluminol(or AEI), aminoethylethylisoluminol (or AEEI), aminobutylisoluminol (orABI), aminobutylethylisoluminol (or ABEI), aminopentylethylisoluminol(or APED, aminohexylisoluminol (or AHI), aminohexylethylisoluminol (orAHEI), aminooctylmethylisoluminol (or AOMI) or aminooctylethylisoluminol(or AOEI), as described in the publication Dodeigne C. et al (2000),Talanta 51, 415-439, “Chemiluminescence as diagnostic tool. A review”.

Developing

The developing step(s) correspond(s) to the detection of the signalproduced by the detection marker(s).

When the detected signal is a fluorescence luminescence signal, the“produced signal” is in particular an “emitted signal”.

The developing step(s) depend(s) on the type of marker used.

The signal produced or emitted by a direct marker of the fluorophoretype can be read directly by fluorescence.

An indirect marker of the enzyme type, the luminescent compound type orthe biotin type requires the addition of a reporter.

As indicated above, an indirect marker of the biotin type requires theaddition of a reporter, preferably a reporter coupled to a marker.

If a reporter is coupled to an indirect marker, for example an enzyme,it is necessary to add, in a later step, a substrate of that indirectmarker, for example a substrate of that enzyme.

As an example, if a reporter is coupled to the peroxidase, it isnecessary to add, in a later step, a substrate of that enzyme, such asluminol.

In one preferred embodiment, a signal detected by chemiluminescence,said signal being produced by a chemiluminescent compound produced bythe reaction of a peroxidase enzyme with its substrate, for exampleluminol, isoluminol and/or a derivative generally also requires thepresence of an oxidizer and, if applicable, an electron mediator.

Generally, the chemiluminescence reaction is done using a kit comprisingat least two solutions.

The first solution comprises the substrate for the peroxidase, forexample the luminol, the isoluminol and/or a derivative of the luminolor the isoluminol, and an electron mediator; the second solutioncomprises an oxidizer. As an example, it is possible to use thefollowing kits: “Immun-star western C” (Bio-Rad, United States),“ELISTAR ETA C Ultra ELISA” (Cyanagen, Italy), “Supersignal West Pico”(Thermo Scientific, United States), “Chemiluminescent Sensitive PlusHRP” (Surmodics, United States).

Solid Support Appropriate for a Secure Multiplex Analysis

The support(s) used to carry out the analysis method according to theinvention are solid supports.

A solid support can be made from any material appropriate to carry outthe analysis method.

A solid support is for example a support with a base of a polymer or amixture of polymers. An appropriate solid support according to theinvention is for example a support made from polystyrene, polypropylene,poly(meth)acrylate, polybutadiene or combinations thereof.

Another type of appropriate solid support according to the invention isfor example an inorganic support, such as glass, and/or a metal support.

A support may be in the form of a plate, a microplate, a slide, beads ora membrane.

Another example of an appropriate solid support is a membrane, forexample a membrane made from nitrocellulose, PVDF (polyvinylidenefluoride), nylon or combinations thereof.

One preferred solid support is made from polystyrene or polypropylene.

Depending on the technology used, the multiplex analysis method can becarried out using a single solid support, for example a solid supportcomprising at least one compartment, said compartment comprising atleast two spots, or on a set of solid supports, for example a set ofbeads.

The controls according to the invention are transposable to use on asingle solid means of the analytes are in the form of spots, and in thesecond case, the controls and the detection means of the analytes are inthe form of beads.

The beads (which may also be called “particles”, “microbeads” or“microparticles”) can be in solution or suspension or fixed on anothersolid support, for example a plate, a microplate, a slide or a membrane,and in particular fixed to the bottom of one or several wells of a solidsupport (for example of a microplate).

Depending on the solid support(s) used, a control of the deposition of asample is called spot to control the deposition of a sample or bead tocontrol the deposition of a sample; a control of the deposition of oneor several detection ligand(s) of an analyte is called spot to controlthe deposition of a detection ligand of an analyte or bead to controlthe deposition of a detection ligand of an analyte; and a control of thedeposition of a reporter is called spot to control the deposition of areporter or bead to control the deposition of a reporter.

In one preferred embodiment, the solid support(s) are appropriate forimplementing a multiplex analysis in the form of an immunological testof the sandwich type.

A solid support according to the invention also has the advantage ofbeing able to detect analytes independently from the matrix of thesample. For example, the detection of analytes present in the blood maybe carried out using a solid support according to the invention from ablood sample or a blood derivative, such as plasma or serum, or amixture of blood and/or blood derivative samples.

Solid Support Appropriate for a Secure Multiplex Analysis on Spots

The present invention particularly relates to a solid supportappropriate for a multiplex analysis of at least one sample, comprisingat least one compartment, said compartment comprising at least onecontrol spot and at least two detection spots for an analyte,characterized in that said control spot is selected from the groupconsisting of a spot for controlling the deposition of a sample, a spotfor controlling the deposition of a detection ligand of an analyte and aspot for controlling the deposition of a reporter.

A solid support comprises at least one compartment (also called analysiszone), preferably at least two compartments.

According to one particular embodiment of the invention, a solid supportcomprises a single compartment. Said single compartment may be acompartment comprising one or several walls. Alternatively, said singlecompartment can have no walls and then be comparable to the solidsupport itself. The bottom of the compartment can then consist of singlecompartment that may or may not include one or several walls is a slideor a membrane.

In one particular embodiment of the invention where a solid support (forexample a slide or a membrane) comprises a single compartment,typically, at least one (for example one or two) solid support is usedper sample to be analyzed.

When a solid support comprises at least two compartments, they areisolated from one another, such that they do not communicate with oneanother, i.e., such that the various compositions or solutions used forthe analysis cannot circulate from one compartment to another during theanalysis. Thus, a solution added into one compartment will not go intothe other compartments. For example, the compartment(s) comprise or aremade up of a bottom and one or several walls, said wall(s) isolating thecompartment(s) from one another such that they do not communicate withone another.

One compartment of the solid support is used per sample to be analyzed.

One example compartment is a well.

A solid support is for example a microplate.

The microplate is typically a microplate with 96 wells or 384 wells.

A compartment of the solid support used to analyze a sample comprises atleast three spots, for example three spots, four spots or five spots, orat least six spots, preferably six spots, seven spots, eight spots, morepreferably at least nine spots, for example nine spots, ten spots,eleven spots, twelve spots, thirteen spots, fourteen spots, fifteenspots, sixteen spots or more than sixteen spots.

Here, a “spot” refers to a zone of a compartment of a solid supportcomprising at least one compound of interest bonded to the surface ofsaid compartment, through noncovalent physicochemical interactions (inparticular of the weak bond type, for example, ionic, van der Waals,hydrogen and/or hydrophobic) and/or by covalent bonds.

A spot may comprise, aside from the compound(s) of interest, at leastone polymer, in particular at least one polymer including hydrophilicgroups, for example at least one hydrogel.

“At least”, within the meaning of the present application, refers to oneor several, several in particular meaning two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, sixteen ormore than sixteen.

A spot corresponds to a well-defined zone, generally spherical or ovaland small, for example comprised between 0.0078 mm² to 5.309 mm²,preferably from 0.196 mm² to 3.142 mm², more preferably comprised from0.503 mm² to 2.011 mm².

A spot may have a discoid, cylindrical or approximately discoid orcylindrical shape, for example oval, in particular when a solid supportis a microplate or a slide.

Alternatively, a spot may have a square or rectangular shape (this mayin particular be a strip), for example when a solid support is amembrane, or any other shape.

The spots are obtained using techniques well known by those skilled inthe art, such as those disclosed in U.S. Pat. No. 7,470,547 B2, U.S.Pat. No. 6,576,295 B2, U.S. Pat. No. 5,916,524 A and U.S. Pat. No.5,743,960 A.

For example, a spot is obtained by depositing at least one drop of asolution containing a determined quantity of said compound(s) ofinterest in a specific location on the surface of the compartment.

When a spot comprises at least one polymer (for example at least onehydrogel), said spot may be obtained by depositing at least one drop ofa solution containing a determined quantity of said compound(s) ofinterest in a specific location on the surface of the compartment onwhich said polymer has been previously deposited.

A spot can also be obtained by in situ synthesis of said compound(s) ofinterest in a specific location on the surface of the compartment. Saidcompound(s) of interest are qualified as probes in this case. This mayinvolve a nucleic acid or a peptide (see for example document U.S. Pat.No. 5,143,854).

The surface of the compartment is also called “solid phase”.

A compound of interest is generally a capture ligand, a carrier moleculecoupled to an indirect marker or an indirect marker. The capture ligand,the carrier molecule coupled to an indirect marker and the indirectmarker are in particular as defined above.

In one advantageous embodiment, each compartment of a solid supportcomprises the same number of spots. Furthermore, each compartment of asolid support comprises the same number of spots and the same spotcomposition.

In another advantageous embodiment, a support may comprise one orseveral compartments without spots, or with a different number of spotsand/or spot composition. Part or all of a support may for examplecomprise at least two separate groups (or types) of spots orcompartments, each of the separate groups having a different number ofspots and/or spot composition.

A compartment comprises at least one control spot (for example, at leastone spot for controlling the deposition of a sample), preferably atleast two control spots, and at least two detection spots for ananalyte.

A compartment generally comprises at least one spot per analyte to bedetected, each analyte for example being able to correspond to aninfection or disease to be detected, the evolution of an infection ordisease, a condition (pathological or not) of the subject, a risk ofdeveloping a condition (pathological or not) or a marker of resistanceto a treatment. Several spots of a compartment may also be intended toanalyze a same analyte.

The spot for controlling the deposition of a sample or for controllingthe deposition of a detection ligand of an analyte preferably comprisesa capture ligand. The capture ligand is in particular as defined above.

A same spot may comprise several different capture ligands (for example,several antibodies and/or antigens), which are generally specific to asame pathology, infection or disease to be detected (in particularspecific to a same virus, a same bacteria, a same fungus or a sameparasite), or specific to a same evolution of an infection or disease, asame condition (pathological or not) of a subject, a same risk ofdeveloping a condition (pathological or not) or a same marker ofresistance to a treatment.

The present invention particularly relates to a solid supportappropriate for a multiplex analysis of at least one sample, comprisingat least one compartment, said compartment comprising at least onecontrol spot and at least two detection spots for an analyte,characterized in that said control spot is a spot for controlling thedeposition of a sample or a spot for controlling the deposition of adetection ligand of an analyte. Said compartment may further comprise atleast one spot for controlling the deposition of a reporter.

In one preferred embodiment, the spot for controlling the deposition ofa sample or for controlling the deposition of a detection ligand of ananalyte comprises one or at least one capture ligand, said captureligand(s) not being a nucleic acid.

More generally, the spot for controlling the deposition of a sample orfor controlling the deposition of a detection ligand of an analytepreferably does not comprise nucleic acid.

In one preferred embodiment, the spot for controlling the deposition ofa sample or for controlling the deposition of a detection ligand of ananalyte comprises one or at least one capture ligand selected from thegroup consisting of an antibody, an antigen, a peptide, a carbohydrateand a lipid.

The present invention particularly relates to a solid supportappropriate for a multiplex analysis of at least one sample, comprisingat least one compartment, said compartment comprising at least twocontrol spots and at least two detection spots for an analyte,characterized in that said control spots are selected from the groupconsisting of a spot for controlling the deposition of a sample, a spotfor controlling the deposition of a detection ligand of an analyte and aspot for controlling the deposition of a reporter.

A solid support according to the invention allows a secure multiplexanalysis.

In one advantageous embodiment, the present invention relates to a solidsupport appropriate for a multiplex analysis of at least one sample,comprising at least one compartment, said compartment comprising atleast two control spots and at least two detection spots for an analyte,characterized in that said control spots are selected from among a spotfor controlling the deposition of a sample and a spot for controllingthe deposition of a detection ligand of an analyte.

The compartment(s) of the solid support may for example comprise atleast two spots for controlling the deposition of a sample or at leasttwo spots for controlling the deposition of a detection ligand of ananalyte.

In one preferred embodiment, the compartment(s) of the solid supportcomprise at least one spot for controlling the deposition of a sampleand at least one spot for controlling the deposition of a detectionligand of an analyte.

As explained above, in case of indirect marking of at least onedetection ligand, it is useful for the compartment(s) to comprise atleast one spot for controlling the deposition of a reporter.

The present invention also relates to a solid support appropriate for amultiplex analysis of at least one sample, comprising at least onecompartment, said compartment comprising at least one spot forcontrolling the deposition of a sample, at least one spot forcontrolling the deposition of a detection ligand of an analyte, at leastone spot for controlling the deposition of a reporter, and at least twospots for detecting an analyte.

The compartment(s) of the solid support may therefore comprise at leasttwo spots for controlling the deposition of a reporter, for example twoor three spots for controlling the deposition of a reporter, foranalyses involving at least two different indirect markers. In such acase, each spot for controlling the deposition of a reporter is specificto a marker.

Set of Beads Appropriate for a Secure Multiplex Analysis

When a solid support is a bead, the multiplex analysis method for asample is done with a set of beads.

The present invention thus also relates to a set of beads appropriatefor a multiplex analysis of a sample, comprising at least one controlbead and at least two detection beads for an analyte, characterized inthat the control bead is selected from the group consisting of a beadfor controlling the deposition of a sample, a bead for controlling thedeposition of a detection ligand of an analyte and a bead forcontrolling the deposition of a reporter.

Within the meaning of the present application, “at least one bead X”means at least one type (or group) of beads X, “bead X” being able tomean “control bead”, “bead for controlling the deposition of a sample”,“bead for controlling the deposition of a detection ligand of ananalyte” or “bead for controlling the deposition of a reporter”.

A “type of beads” (or “group of beads”) within the meaning of theinvention comprises or consists of several beads (for example 10, 50,100, 200, 300 or 500 beads), which are identical to one another or atleast several beads on the surface of which the same compound(s) ofinterest is (are) fixed.

Similarly, “at least two beads X” respectively means at least two beadsX or two different types (or “groups”) of beads X, “bead X” being ableto mean “control bead”, “bead for detecting an analyte”, “bead forcontrolling the deposition of a sample”, “bead for controlling thedeposition of a detection ligand of an analyte” or “bead for controllingthe deposition of a reporter”.

Each bead is also covered with at least one compound of interest bondedto the surface of the bead, also called “solid phase”.

A set of beads used to analyze a sample comprises at least three beads(or types of beads), for example three, four or five beads (or types ofbeads), or at least six beads (or types of beads), preferably six,seven, eight beads (or types of beads), more preferably at least ninebeads (or types of beads), for example nine, ten, eleven, twelve,thirteen, fourteen, fifteen beads or sixteen beads (or types of beads),or more than sixteen beads (or types of beads).

In the present application, “bead”, “particle”, “microbead” or“microparticle” means any particle, preferably spherical orapproximately spherical, with a size that may be comprised from 0.3 μmto 100 μm in diameter, preferably from 0.5 μm to 40 μm. Such particlesare for example manufactured by the companies Luminex, Merck and Dynal.A bead able to be used in the context of the present invention may alsobe a particle in the form of a cube or slab or approximately in the formof a cube or slab, the length of the sides of which would for example becomprised from 0.3 μm to 100 μm, preferably from 0.5 μm to 40 μm.

A bead according to the invention is preferably made up of one orseveral polymers that are inert relative to the components of thebiological samples; it is solid and insoluble in the samples to beanalyzed. Examples of inert polymers that may be used are a polyester,polyether, polyolefin, polyamide, polysaccharide, polyurethane orcellulose.

One or several functional groups may be incorporated with said inertpolymer(s) to allow the fixing or coupling of one or several compoundsof interest (for example proteins, peptides, glycoproteins, lipids,carbohydrates or nucleic acids). These functional groups, known by thoseskilled in the art, may be selected from the group consisting of amine(═NH2) functions or ammonium functions (—NH3+ or —NHR, R representing analiphatic chain, preferably an:

-   -   alkyl chain from 1 to z carbon atoms, linear or branched        (substituted or not), z preferably being an integer comprised        from 1 to 20,    -   alkenyl chain from 2 to z carbon atoms, linear or branched, z        preferably being an integer comprised from 1 to 20, or    -   an aryl radical),        alcoholic functions (—OH), carboxylic functions (—COOH),        isocyanate functions (—NCO), thiol functions (SH) or epoxy        functions. The most commonly used monomers to introduce —COOH        carboxylic functions into the polyolefins are acrylic acid or        methacrylic acid.

A bead is in fact covered with one or several compounds of interest,using any appropriate method well known by those skilled in the art. Thefixing of the compound(s) of interest on the surface of a bead may bedone by electrostatic attraction, affinity interaction, hydrophobicinteraction and/or covalent coupling.

In one preferred embodiment, the compound(s) of interest are fixed tothe surface of the bead by covalent coupling.

The methods for fixing one or several compounds to the surface of a beadare well known by those skilled in the art (cf. for example LUMINEXxMAP® Antibody Coupling KitUser Manual, the article by Joseph Dasso etal. (Journal of Immunological Methods 263 (2002) 23-33) or documentWO1997/014028).

For example, in the case of the bead comprising carboxyl chemicalfunctions on its surface, these chemical functions may be converted intoan activated ester formed by a reaction with the1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride andN-hydroxysulfosuccinimide. A compound of interest, such as an antibody,a protein or a peptide, may then be grafted, by a free amine grouppresent on said compound of interest, on the activated ester groups ofeach bead. The conversion into an activated ester form and the couplingof the compound of interest on a bead are done using procedures wellknown by those skilled in the art (cf. for example U.S. Pat. No.7,141,362).

The beads or part of the beads of a set of beads are preferably magneticbeads, such as beads using the Luminex xMAP® technology, in order toallow the recovery of the beads between the different steps of themethod, and in particular at the end of the washing steps.

Each bead is marked with a code so as to be able to differentiate itfrom the other beads of the set of beads, for example a fluorescencecode or a barcode.

The marking of a bead with a code, for example by fluorescence or abarcode, can be done using any appropriate method well known by thoseskilled in the art, for example as described in documents EP 1,802,710and EP 1,049,807.

As for a solid support comprising at least one compartment, a compoundof interest is generally a capture ligand, a carrier molecule coupled toan indirect marker or an indirect marker. The capture ligand, thecarrier molecule coupled to an indirect marker and the indirect markerare in particular as defined above.

The set of beads according to the invention comprises one bead (or typeof beads) per desired control and at least one bead (or at least onetype of beads) per analyte to be detected. Several beads (or types ofbeads) can also be intended to detect a same analyte.

The bead for controlling the deposition of a sample or for controllingthe deposition of a detection ligand of an analyte preferably comprisesa capture ligand. The capture ligand is in particular as defined above.

A same bead (or each bead of a given type of beads) may be covered withseveral different capture ligands (for example, several antibodiesand/or antigens), which are generally specific to a same infection to bedetected, and in particular specific to a same virus, a same bacteria ora same parasite.

The set of beads therefore comprises at least one control bead (forexample, at least one bead for controlling the deposition of a sample),preferably at least two control beads, and at least two detection beadsfor an analyte.

At least one set of beads is used per sample to be analyzed.

The present invention particularly relates to a set of beads appropriatefor a multiplex analysis of at least one sample, comprising at least onecontrol bead and at least two detection beads for an analyte,characterized in that said control bead is a bead for controlling thedeposition of a sample or a bead for controlling the deposition of adetection ligand of an analyte. Said set of beads may further compriseat least one bead for controlling the deposition of a reporter.

In one preferred embodiment, the bead for controlling the deposition ofa sample or for controlling the deposition of a detection ligand of ananalyte comprises one or at least one capture ligand, said captureligand(s) not being a nucleic acid.

More generally, the bead for controlling the deposition of a sample orfor controlling the deposition of a detection ligand of an analytepreferably does not comprise nucleic acid.

In one preferred embodiment, the bead for controlling the deposition ofa sample or for controlling the deposition of a detection ligand of ananalyte comprises one or at least one capture ligand selected from thegroup consisting of an antibody, an antigen, a peptide, a carbohydrateand a lipid.

The present invention particularly relates to a set of beads appropriatefor a multiplex analysis of a sample, comprising at least two controlbeads and at least two detection beads for an analyte, characterized inthat said control beads are selected from the group consisting of a beadfor controlling the deposition of a sample, a bead for controlling thedeposition of a detection ligand of an analyte and a bead forcontrolling the deposition of a reporter.

In one advantageous embodiment, the present invention relates to a setof beads appropriate for a multiplex analysis of a sample, comprising atleast two control beads and at least two detection beads for an analyte,characterized in that said control beads are chosen from among a beadfor controlling the deposition of a sample and a bead for controllingthe deposition of a detection ligand of an analyte.

The set of beads may for example comprise at least two beads forcontrolling the deposition of a sample or at least two beads forcontrolling the deposition of a detection ligand of an analyte.

In one preferred embodiment, the set of beads comprises at least onebead for controlling the deposition of a sample and at least one beadfor controlling the deposition of a detection ligand of an analyte.

As explained above, in case of indirect marking of at least onedetection ligand for an analyte, it is advantageous to use at least onebead for controlling the deposition of a reporter.

The present invention also relates to a set of beads appropriate for amultiplex analysis of a sample, comprising at least one bead forcontrolling the deposition of a sample, at least one bead forcontrolling the deposition of a detection ligand of an analyte, at leastone bead for controlling the deposition of a reporter and at least twobeads for detecting an analyte.

The set of beads may therefore comprise at least two beads forcontrolling the deposition of a reporter, for example two or three beadsfor controlling the deposition of a reporter, for analyses involving atleast two different indirect markers. In such a case, each bead forcontrolling the deposition of a reporter is preferably specific to amarker.

The set of beads according to the invention allows a secure multiplexanalysis.

Control of the Deposition of a Sample

The control of the deposition of the sample makes it possible to verifythat the sample has been placed in the presence of the spots of acompartment of the solid support or with a set of beads.

The control of the deposition of a sample comprises at least one captureligand, in particular an antibody or an antigen, specific to a controlcompound naturally present in the sample to be analyzed. The controlcompound is in particular as defined above.

Thus, a signal detected at the control of the deposition of a samplemakes it possible to validate the deposition step of the sample.

The deposition of a sample consists of adding a sample to be analyzedinto a compartment of the solid support according to the inventioncomprising at least one control spot and at least two analyte detectionspots, or placing the sample in the presence of at least one set ofbeads according to the invention.

A control compound then fixes to the capture ligand at the sampledeposition control. A control compound thus fixed can be detected by aspecific detection ligand of the control compound.

Preferably, a detection ligand of a control compound is specific to azone of the control compound remote from the zone of the controlcompound to which the capture ligand used specifically bonds, so as toavoid competition of the capture ligand and the detection ligand withrespect to the control compound, due to a steric hindrance.

When a control compound is a complex of at least two molecules, thedetection ligand or one of the detection ligands can be specific to oneof the two molecules and the capture ligand or one of the captureligands can be specific to the other molecule of the complex.

Alternatively, when a control compound is a complex of at least twomolecules, the detection ligand or one of the detection ligands and thecapture ligand or one of the capture ligands can be specific to the samemolecule, and even the same zone of the molecule, when the complexcomprises at least two of these identical molecules, thereby allowingthe simultaneous fixing of the detection antibody and the captureantibody on the complex.

For example, when a control compound is the soluble receptor of thetransferrin complexed to the transferrin, a deposition control maycomprise, as capture ligand, a specific antibody either of thetransferrin, or of the soluble receptor of the transferrin. Preferably,the or one of the capture ligand(s) is a specific antibody of thesoluble receptor of the transferrin, in order to avoid interference withthe free transferrin that may be found in a blood sample.

In one preferred embodiment, the or one of the capture ligand(s) of thedeposition control is specific to the soluble receptor of thetransferrin and the or one of the detection ligand(s) of the or acontrol compound is either specific to the transferrin, or specific tothe soluble receptor of the transferrin.

In certain embodiments, a control of the deposition of a sample alsomakes it possible to verify that one or several analyte detectionligand(s) have been placed in the presence of the spots of a compartmentof a solid support or of a set of beads, when said analyte detectionligand(s) are not added at the same time as sample to be analyzed, butfor example in a later step, at the same time as the addition of thedetection ligand(s) of the control compound. “At the same time as theaddition of the detection ligand(s) of the control compound” generallymeans that said analyte detection ligand(s) are added in the form of asolution comprising said analyte detection ligand(s) and the detectionligand(s) of the control compound.

Alternatively, said analyte detection ligand(s) and said detectionligand(s) of the control compound can be added in the same step, but inthe form of separate solutions.

Indeed, when a sample to be analyzed comprises a compound that couldinterfere with the detection ligand of the control compound, it may benecessary to add the detection ligand to the control compound in a stepafter the deposition step of the sample, in particular after thedeposition step of the sample followed by at least one washing step. Inthis case, all or part of the detection ligand(s) of the analytes can beadded at the same time as the detection ligand of the control compound.The control of the deposition then also makes it possible to validatethat these detection ligands have been placed in the presence of thespots of a compartment of the solid support or of the set of beads.

For example, a blood sample comprises both the soluble receptor of thetransferrin complexed to the transferrin and the free transferrin, i.e.,not bonded to the receptor. Thus, if the or one of the detectionligand(s) is a specific antibody of the transferrin and it is added atthe same time as the sample, it would risk bonding in part to the freetransferrin, and the detection of the control compound would bedistorted. In such a case, said detection ligand of the control compoundis contributed in a subsequent step, i.e., after the deposition step ofthe sample followed by at least one washing step.

In one particular embodiment, the control(s) of the deposition of thesample do not comprise a specific antibody of factor XIII (i.e., neithera specific antibody of the tetrameric form, nor a specific antibody of asubunit of factor XIII). Preferably, the control(s) of the deposition ofa sample do not comprise a specific capture ligand of factor XIII (i.e.,neither a specific capture ligand of the tetrameric form, nor a specificcapture ligand of a subunit of factor XIII).

In one particular embodiment, the control(s) of the deposition of asample do not comprise a specific capture ligand (in particular aspecific antibody) of an IgG, an IgM or more generally an immunoglobulin(in particular a human IgG, a human IgM or a human immunoglobulin).

It may be very advantageous to use at least two controls of thedeposition of a sample, these controls detecting different controlcompounds and/or a same control compound but with differentsensitivities, for example when the concentration of the controlcompound may vary, for example based on the nature of the sample and/orthe condition of the subject from which the sample was taken, it may beadvantageous to use at least two different control compounds.

Control of the Deposition of a Detection Ligand of an Analyte

The control of the deposition of a detection ligand of an analyte makesit possible to verify that one or several analyte detection ligand(s)deposited at the same time as the detection ligand of an additive and/orone or several analyte detection ligand(s) deposited at the same time asan additive have been placed in the presence of the spots of acompartment of a solid support or of a set of beads.

“Deposited at the same time as the detection ligand of an additive”generally means that said analyte detection ligand(s) are added in theform of a solution comprising said analyte detection ligand(s) and thedetection ligand(s) of an additive. Alternatively, said analytedetection ligand(s) and/or the detection ligand(s) of an additive can beadded in the same step, but in the form of separate solutions.

“Deposited at the same time as an additive” generally means that saidanalyte detection ligand(s) are added in the form of a solutioncomprising said analyte detection ligand(s) and an additive.Alternatively, said analyte detection ligand(s) and/or one or severaladditives can be added in the same step, but in the form of separatesolutions.

The control of the deposition of a detection ligand of an analyte alsomakes it possible to verify the incubation and washing steps. Indeed,the concentration of the additive placed in the presence of the spots ofa compartment of the solid support or of a set of beads being known, avariation of the detected signal makes it possible to identify adeficiency of the method at the steps of the method, and in particularthe incubation and washing steps. This may in particular made itpossible to avoid “false positives” resulting from deficient incubationand/or washing steps, i.e., not done or done poorly.

A “false positive” is a positive result reflecting the presence of oneor several analytes to be detected in a sample, whereas said analyte(s)were not present in the sample and therefore should not have beendetected.

The control of the deposition of a detection ligand of an analytecomprises at least one specific capture ligand of an additive that isnot present in the sample to be analyzed and that is used during theimplementation of the multiplex analysis method. An additive is inparticular as defined above.

Thus, a signal detected at the control of the deposition of a detectionligand of an analyte makes it possible to validate the deposition stepof the analyte detection ligand(s) deposited at the same time as thedetection ligand of an additive.

An additive placed in the presence of the spots and/or of a set of beadsfixes to the capture ligand at the control of the deposition of adetection ligand of an analyte. An additive thus fixed can be detectedby a specific detection ligand of said additive.

For example, when an additive comprises digoxigenin, a capture ligand ofthe method control may be a specific antibody of the digoxigenin.Preferably, a detection ligand of an additive is also a specificantibody of digoxigenin.

It may be advantageous to use several different controls of thedeposition of a detection analyte, and therefore an equal number ofdifferent additives, for example if the detection ligands of theanalytes are added in several different steps, in particular during atleast three different steps.

According to one particular embodiment of the invention,

-   -   a control compound that fixes to a capture ligand of a        deposition control of the sample during the implementation of        the analysis method according to the invention is detected by a        specific detection ligand of said control compound        (immunological format of the sandwich type), and    -   an additive that fixes to a capture ligand of a deposition        control of a detection ligand of an analyte during the        implementation of the analysis method is detected by a specific        detection ligand of said additive (immunological format of the        sandwich type).

These sandwich-type immunological formats make it possible to achieve ahigh level of specificity and/or sensitivity, compared to otherimmunological formats, and in particular indirect formats.

Control of the Deposition of a Reporter

The control of the deposition of a reporter makes it possible to verifythat the detection marker reporter has been placed in the presence ofthe spots of a compartment of a solid support or of a set of beads.

The control of the deposition of a reporter is only useful in case ofindirect marking of at least one detection ligand of an analyte.

The control of the deposition of a reporter comprises an indirect markeror a carrier molecule coupled to an indirect marker.

The indirect marker of the control of the deposition of a reporter isidentical to the indirect marker of at least one detection ligand of ananalyte.

If at least two different indirect markers are used to detect at leasttwo analyte detection ligands, it is preferable to use a control of thedeposition of a reporter for each marker.

In one advantageous embodiment according to the invention, a single andsame indirect marker is used to mark the detection ligand(s), andtherefore a single control of the deposition of a reporter can be used.

Thus, a signal detected at the control of the deposition of a reportermakes it possible to validate the deposition step of the reporter of thecorresponding detection marker.

The reporter thus fixes to the detection marker at the control of thedeposition of a reporter and a signal is obtained, if necessary afteradding the substrate of the marker of said reporter, in case of positivecontrol of the deposition of a reporter.

For example, when the indirect marker is biotin, the control of thedeposition of a reporter comprises biotin or a carrier molecule coupledto biotin.

Secure Multiplex Analysis Method

The present invention thus relates to a multiplex analysis method fordetecting at least n analytes in at least one sample, n being an integergreater than or equal to 2, said method comprising at least steps a), c)and e), at least steps a), c) and f) or at least steps a), b), c) and d)as follows:

-   -   a) providing at least one solid support comprising at least one        compartment as defined above or at least one set of beads as        defined above,    -   b) placing, in the presence of the spots of said compartment or        said set of beads: l detection ligands of p analytes to be        detected, and, if applicable, at least one additive, l        preferably being greater than or equal to p,    -   c) placing a sample to be analyzed in the presence of the        spot(s) of said compartment or of said set of beads,    -   d) placing, in the presence of the spots of said compartment or        of said set of beads:        -   l′ detection ligands of m analytes to be detected, and, if            applicable, at least one detection ligand of said            additive(s), l′ preferably being greater than or equal to m,    -   e) placing, in the presence of the spots of said compartment or        of said set of beads:        -   at least one detection ligand of a control compound and l″            detection ligands of y analytes to be detected, l″            preferably being greater than or equal to y, and    -   f) optionally placing at least one reporter in the presence of        the spots of said compartment or of said set of beads,    -   g) optionally, in particular when said or one of said reporters        of step f is coupled to a marker, in particular an indirect        marker (for example, an enzyme), placing at least one second        reporter of said marker coupled to the reporter of step f) (for        example, a substrate, such as luminol, isoluminol or one of        their derivatives) in the presence of the spots of said        compartment or said set of beads,    -   l, l′, l″, m, p and y being integers greater than or equal to 0        and the sum m+p+y being greater than or equal to 1.

A method for detecting at least n analytes may consist of detecting adifferent number of analytes that is less than n, when at least twoanalytes to be detected are identical. For example, at least two spotsof a compartment of a solid support or at least two beads (or types ofbeads) of a set of beads are intended to detect a same analyte.

The number “n” is generally equal to the number of detection spots of ananalyte of a compartment of a solid support or the number of beads (ortypes of beads) for detecting an analyte of a set of beads.

Preferably, the sum m+p+y is comprised from 1 to n.

When several analytes to be detected are identical, it is possible touse a same detection ligand for at least two or all of the identicalanalytes. The sum m+p+y may then be greater than or equal to 1 andstrictly less than n.

In another embodiment, the sum m+p+y is equal to n, for example whenthere are n detection spots of an analyte in a compartment of a solidsupport or n beads (or types of beads) for detecting an analyte of a setof beads.

As outlined below, steps a) to e) can be done simultaneously and/orsuccessively (for example, some of these steps may be donesimultaneously, while others are done successively), at least for two ofthem in the order a) to e) or in another order, step a) always being thefirst step carried out; when the method [comprises] step f) andoptionally step g), these steps are always carried out after steps a) toe).

When the method comprises at least steps a), c) and e), a solid supportcomprises at least one compartment, said compartment comprising at leastone spot for controlling the deposition of a sample, or a set of beadscomprises at least one bead for controlling the deposition of a sample.

When the method comprises at least steps a), c) and f), a solid supportcomprises at least one compartment, said compartment comprising at leastone spot for controlling the deposition of a reporter, or a set of beadscomprises at least one bead for controlling the deposition of areporter.

When step b) comprises placing the spots or the set of beads in thepresence of at least one additive, step d) preferably comprises placingthe spots or the set of beads in the presence of at least one detectionligand of said additive(s).

When the method comprises at least steps a), b), c) and d), at least oneadditive being placed in the presence of the spots or of the set ofbeads in step b) and at least one detection ligand of said additive(s)being placed in the presence of the spots or the set of beads in stepd), a solid support comprises at least one compartment, said compartmentcomprising at least one spot for controlling the deposition of adetection ligand of an analyte, or a set of beads comprises at least onebead for controlling the deposition of a detection ligand of an analyte.

When the method comprises steps a), b), c) and d), but does not comprisestep e), at least one additive is placed in the presence of the spots orof the set of beads in step b) and at least one detection ligand of saidadditive(s) is placed in the presence of the spots or the set of beadsin step d).

The expression “place a compound X in the presence of the spots of acompartment” means that the compound X is added into a compartmentcomprising said spots, said compartment being intended to analyze asample.

The expression “place a compound X in the presence of a set of beads”means that the compound X is placed in the presence of at least one setof beads intended to analyze a sample in any appropriate containercontaining said set of beads. One example of an appropriate container isa microtube, a microplate or a reaction dish.

When at least two of steps b) to f) are done simultaneously, thecompound(s) X may be placed separately in the presence of the spot(s) ofa compartment or of the bead(s) or of the set(s) of beads intended toanalyze a sample, i.e., in the form of separate compositions.Alternatively, these compounds X or some of these compound(s) X may beplaced in the presence of the spot(s) of a compartment or of the bead(s)or of the set(s) of beads intended to analyze a sample in the form ofone or several mixtures.

The different compounds are placed in the presence of spots of eachcompartment or each set of beads for a certain length of time, forexample from 1 second to 2 hours, preferably 1 minute to 1 hour, morepreferably 5 minutes to 50 minutes, still more preferably from 10minutes to 40 minutes.

One skilled in the art knows how to determine the appropriatetemperature for each incubation step. The temperature of an incubationmay for example be 4° C., a temperature comprised from 19° C. to 24° C.,37° C. or 40° C.

The different components used during steps b), d), e), f) and g) arewell known by those skilled in the art. They for example make itpossible to form antigen-antibody, marker-reporter complexes.

Step a) consists of providing a solid support as defined abovecomprising at least one compartment or at least one set of beads asdefined above.

Step a) in particular means that the multiplex analysis method isimplemented using said solid support or said set of beads, i.e., usingsaid solid support or said set of beads.

The multiplex analysis method according to the invention is thereforeimplemented either by using a solid support with compartment(s), forexample of the microplate type, or with a set of beads.

For x samples to be analyzed, step a) consists of providing:

-   -   a solid support comprising at least x compartments or several        solid supports so as to have at least x compartments with all of        the solid supports (for example, when a solid support includes        only one compartment, which may, in one particular embodiment,        be likened to the solid support itself, providing at least x        solid supports), or    -   at least x sets of beads.

The multiplex analysis method according to the invention for examplemakes it possible to detect n analytes in at least 1 sample, at leasttwo samples, at least 5 samples, at least 10 samples, preferably atleast 20 samples, for example at least 40 samples, at least 60 samplesor at least 80 samples.

The number n is an integer greater than or equal to 2, for example 2, 3,4, 5, preferably greater than or equal to 6, for example 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18 or greater than 18.

When the steps are not done simultaneously, they are generally followedby one or several washing steps.

A washing step makes it possible to eliminate the compounds not bondedto the spots or beads or to the various compounds bonded to said spotsor beads.

Typically, a washing step consists of at least one cycle, preferably atleast two cycles, more preferably 3 to 6 cycles, for distributing (forexample, a volume of 400 μl) and aspirating a wash solution in eachcompartment or in the presence of each set of beads. Typically, thewashing solution may be a Tris NaCl 0.01 M buffer, pH 7.4 doped withTween 20 at 0.1%.

When the method is implemented with at least one solid supportcomprising at least two compartments, the compartments of the solidsupport or supports are preferably identical, in particular in terms ofnumber and composition of the spots.

Alternatively, the method may be implemented with at least one solidsupport comprising at least two separate groups (or types) ofcompartments, each of the separate groups having a different number ofspots and/or spot composition.

When the method is implemented with at least two sets of beads, the setsof beads can be identical, in particular in terms of number andcomposition of the beads.

Alternatively, the method may be implemented with at least two separategroups (or types) of sets of beads, in particular each of the separategroups of sets of beads having a different number and/or composition ofbeads.

Steps b), d), e) and f) are carried out in each compartment intended toanalyze a sample or in each container containing a set of beads intendedto analyze a sample.

Regarding step c), one sample is added per compartment or per containercontaining a set of beads.

The sample(s), the analytes, the additive(s), the control compound(s),the detection ligands of the analytes, the detection ligand(s) of theadditive(s), the detection ligands of the control compound(s) and thereporter(s) are in particular as defined above in the correspondingparagraphs.

An additive used in the method according to the invention is an additiverecognized by the capture ligand(s) of the deposition control of adetection ligand of an analyte.

A control compound present in the sample is a control compoundrecognized by the capture ligand(s) of the deposition control of asample.

Each detection ligand of an analyte is specific to an analyte recognizedby at least one capture ligand present in a detection spot of an analyteor on a detection bead of an analyte.

When step b) is not carried out simultaneously with one of steps c), d)or e), one or several washing steps can be carried out after step b) andbefore said steps c), d) and e) only when l is equal to 0.

When steps b), c), d) and e) are present, they can be carried out in anydesired order: successively, or one, two or three of these stepssimultaneously. However, when a step b) and a step d) respectivelyimplement at least one additive and at least one detection ligand ofsaid additive, said step d) is still done at the same time as or aftersaid step b). Likewise, when a step e) is present, it is always done atthe same time as or after step c), or before step c) as long as there isno washing step between steps e) and c). Furthermore, when step c) isdone after a step b) in which l is greater than or equal to 1 and/orafter a step d) in which l′ is greater than or equal to 1 and/or afterstep e), no washing step is done between this or these steps and stepc).

As an example, when steps b) to e) are present, they can be donesimultaneously and/or successively, in the following orders: b) to e),b) c) e) d), c) b) d) e), c) b) e) d) or c) e) b) d).

Steps c), d) and/or e) can be done simultaneously with step b).

In one advantageous embodiment according to the invention, steps b) andc) are done simultaneously.

In another advantageous embodiment according to the invention, steps c)and d) are done simultaneously. It may indeed be advantageous to placethe sample simultaneously in the presence of the detection ligands ofthe analytes, which interact better in solution with the analyte towhich they are specific than when the analyte is already bonded to thecapture antibody of the analyte.

In still another advantageous embodiment according to the invention,steps b), c) and d) are done simultaneously.

When several additives are used, the method may include one or severalsteps b) (and one or several steps d)), each of steps b) being able tobe done independently before, after or at the same time as step c).There may for example be at least two steps b), one being done before orat the same time as step c), the other being done after or at the sametime as step c).

The compounds of a given step may be provided in the form of a mixture,i.e., a solution, that comprises all of the compounds of that step andoptionally the compounds of one or several steps done simultaneously.

When two or more than two steps are done simultaneously, the compoundsof these different steps may be contributed in the form of one orseveral mixtures, i.e., one or several solutions, preferably in the formof a single mixture (i.e., a single solution).

For example, when steps b), c) and d) are done simultaneously, theadditive(s) (when they are present), the l detection ligands of each ofthe p analytes to be detected, the detection ligand(s) of saidadditive(s) (when they are present), the l″ detection ligands of each ofthe m analytes to be detected can be contributed in the form of a singlemixture.

In one advantageous embodiment, l′ is equal to n, i.e., at least ndetection ligands of the n analytes are added in step d).

In certain embodiments, step e) is done after step c). This inparticular involves the case where the presence of the or one of thedetection ligands for a compound controls whether the sample riskscausing interference at the same time.

For example, when the detection ligand of the control compound is aspecific antibody of the transferrin and the sample comprises freetransferrin, step e) is carried out after step c).

When step e) is carried out after step c), preferably at least onewashing step of the compartments is done after step c) and before stepe).

When the method according to the invention comprises step f), this stepis always done after steps a) to e) and it is generally preceded by atleast one washing step of the spots of each compartment or each set ofbeads.

The method may also comprise several steps f) if at least two depositioncontrols of a reporter are used, i.e., preferably one step f) perreporter.

When the method according to the invention comprises step g), this stepis always done after a step f) and it is generally preceded by at leastone washing step of the spots of each compartment or each set of beads.

The or at least one of the reporters of step f) is preferably both areporter of an indirect detection marker coupled to at least onedetection ligand of an analyte and that of a detection marker present inat least one control of the deposition of a reporter.

It may be advantageous for the controls used for the implementation ofthe multiplex analysis method according to the invention to mimic thedifferent steps of the detection of analytes. For example, if thecapture ligands of the analytes and the detection ligands of theanalytes are antibodies, the detection ligand of the additive and,preferably, the detection ligand of the control compound are alsoantibodies. Likewise, the detection ligand of the additive and,preferably, the detection ligand of the control compound are marked withthe same marker as that of the detection ligands of the analytes.

The method according to the invention generally comprises a step h) fordetecting the signal corresponding to the detection markers of thecontrol(s) (in particular the control spot(s) or the control bead(s))and of the analyte(s).

A control is positive if a positive signal is detected at the end of themethod.

A control is negative if no positive signal is detected at the end ofthe method.

The detection of a positive signal for each control used during theimplementation of the method makes it possible to validate the entiremethod.

The absence of a signal at one or several controls means that one orseveral steps have not taken place correctly. In this case, the obtainedresults must not be used.

When a detection ligand of a control compound is deposited after thedeposition of the sample and after at least one washing step, a negativecontrol of the deposition of a sample means that the sample has not beendeposited and/or that said detection ligand of a control compound hasnot been deposited.

The control of the deposition of a sample, the control of the depositionof a detection ligand of an analyte and, if applicable, the control ofthe deposition of a reporter are complementary and make it possible toensure the proper performance of each of the steps of the method andmake it possible to understand the origin of any flaw encountered in theanalysis method.

The method according to the invention may also comprise:

-   -   at least two steps b) and at least two steps d), when at least        two controls of the deposition of a detection ligand of an        analyte are used,    -   at least two steps f), when at least two controls of the        deposition of a sample are used, and/or    -   at least two steps f), when at least two controls of the        deposition of a reporter are used.

Use of One or Several Controls to Secure a Multiplex Analysis Method ofa Sample.

The present invention also relates to the use of at least one control ofthe deposition of a sample and/or at least one control of the depositionof a detection ligand of an analyte, to secure a multiplex analysis of asample.

Said use may further comprise the use of at least one control of thedeposition of a reporter.

The present invention particularly relates to the use of at least onecontrol selected from the group consisting of a control of thedeposition of a sample, a control of the deposition of a detectionligand of an analyte and a control of the deposition of a reporter, tosecure a multiplex analysis of a sample.

Here, “secure a multiplex analysis of a sample” means guaranteeing thereliability of the results of a multiplex analysis, in particular byavoiding the presence of “false negatives”.

A “false negative” is a negative result reflecting the absence of one orseveral analytes to be detected in a sample, whereas said analyte(s)were present in the sample and should have been detected.

The present invention also relates to the use of at least two controlsselected from the group consisting of a control of the deposition of asample, a control of the deposition of a detection ligand of an analyteand a control of the deposition of a reporter, to secure a multiplexanalysis of a sample.

The present invention particularly relates to the use of at least onecontrol of the deposition of a sample, at least one control of thedeposition of a detection ligand of an analyte and at least one controlof the deposition of a reporter, to secure a multiplex analysis of asample.

Said use is preferably done on a solid support comprising at least onecompartment as defined above or on at least one set of beads as definedabove.

The control of the deposition of a sample, the control of the depositionof a detection ligand of an analyte, the control of the deposition of areporter and the sample are in particular as defined above.

The present invention also relates to a kit, characterized in that itcomprises or consists of at least one solid support according to theinvention and/or at least one set of beads according to the inventionand, if applicable, at least one composition or solution to be used tocarry out a multiplex analysis method according to the invention and/oruser instructions.

Other features and advantages of the invention will better emergethrough the following examples, provided as an illustration andnon-limitingly. These examples and figures illustrate the inventionwithout limiting its scope.

FIGURES

FIG. 1: Example of a solid support of the microplate type for a securemultiplex analysis. A shows a block diagram of a microplate including 18wells. B diagrammatically shows the bottom of a well of the microplate.Each well of the microplate comprises 9 spots, 6 spots of which are eachintended to detect one or several analytes making it possible todiagnose an infection (respectively A1, A2, A3, A4, A5 and A6) and 3control spots: SDC (Sample Deposition Control), PVC (analyte detectionligand deposition control) and RVC (Reporter Deposition Control). Cdiagrammatically shows the three control spots comprising the detectionantibodies bonded to the solid phase (PS). Ac CC: Detection antibody ofthe control compound. Ac Add: Detection antibody of the additive. MS+B:Carrier molecule marked with biotin.

FIG. 2: Diagram of a positive control of the deposition of a sample(SDC) in heterologous format. The control of the deposition of a samplecomprises the capture antibody of the control compound fixed to thesolid phase (PS). The capture antibody here is specific to the solublereceptor of the transferrin (sTfR). The control compound, here complex2:2 soluble transferrin receptor (sTfR):transferrin (Tf) is fixed to thecapture antibody at the control of the deposition of a sample and to thedetection antibody (Ac 2) that is specific to the transferrin. Thedetection antibody (Ac 2) is marked with biotin (B). The developing isdone by adding streptavin coupled to a peroxidase (S-POD), then thesubstrate of this enzyme (not shown). The capture antibody and thedetection antibody can be monoclonal or polyclonal antibodies. In allcases, it involves a heterologous sandwich.

FIG. 3: Diagram of a positive control of the deposition of a sample(SDC) in homologous format. The control of the deposition of a samplecomprises the capture antibody of the control compound fixed to thesolid phase (PS). The capture antibody here is specific to the solublereceptor of the transferrin (sTfR). The control compound, here complex2:2 soluble transferrin receptor (sTfR):transferrin (Tf) is fixed to thecapture antibody at the control of the deposition of a sample and to thedetection antibody (Ac 2) that is also specific to the solubletransferrin receptor (sTfR). The detection antibody (Ac 2) is markedwith biotin (B). The developing is done by adding streptavin coupled toa peroxidase (S-POD), then the substrate of this enzyme (not shown). Thecapture antibody and the detection antibody are for example monoclonalantibodies. In involves a homologous sandwich.

FIG. 4: Diagram of a positive control of the deposition of a detectionligand of an analyte (PVC) The method control comprises the captureantibody of the additive fixed to the solid phase (PS). The captureantibody here is specific to digoxigenin (Dig). The additive comprisesdigoxigenin and BSA, the digoxigenin being complexed to the BSA. Theadditive is bonded via the digoxigenin to the capture antibody at thecontrol of the deposition of a detection ligand of an analyte and to thedetection antibody (Ac 2). The detection antibody (Ac 2) is marked withbiotin (B). The developing is done by adding streptavin coupled to aperoxidase (S-POD), then the substrate of this enzyme (not shown). Thecapture antibody and the detection antibody can both be monoclonalantibodies or both polyclonal antibodies, in which case it involves ahomologous sandwich. The capture antibody can be a monoclonal antibodyand the detection antibody a polyclonal antibody, or vice versa, inwhich case it involves a heterologous sandwich.

FIG. 5: Diagram of a positive control of the deposition of a reporter(RVC). The control of the deposition of a reporter comprises a carriermolecule (MS) coupled to biotin (B) fixed to the solid phase (PH). Thedeveloping is done by adding streptavin coupled to a peroxidase (S-POD),then the substrate of this enzyme (not shown).

FIG. 6: “Spotting” grid situated at the bottom of a well of a microplateand including the spots of 5 analytes to be assayed (A1 to A5) and thethree control spots SDC, PVC and RVC.

FIG. 7: Distribution of the signal of the SDC control beads in simplexformat of a population of 94 samples. The Y-axis shows the number ofsamples and the X-axis shows intervals of Relative FluorescenceIntensity (RFI).

FIG. 8: Distribution of the signal of the PVC control beads in simplexformat of a population of 38 samples. The Y-axis shows the number ofsamples and the X-axis shows intervals of Relative FluorescenceIntensity (RFI).

EXAMPLES Example 1 “Liquid Chip” Method Principle

The two controls described in this example make it possible to validate(cf. table 1):

-   -   for the SDC (“Sample Deposit Control” or “Control of the        deposition of a sample”): the deposition of the sample and also        step 2 (deposition of the conjugates 2, i.e., the detection        ligands deposited in step 2) and step 3) (deposition of the S-PE        reporter: streptavidin coupled to Phycoerythrin), and    -   for the PVC (“Process Verification Control” or “control of the        deposition of a detection ligand of an analyte”): the deposition        of step 1 (deposition of the conjugates 1, i.e., the detection        ligands deposited in step 1) and also step 2 (deposition of the        conjugates 2, in dotted lines because not illustrated in this        example) and step 3) (deposition of the S-PE reporter).

TABLE 1 Steps controlled by the SDC and PVC controls Conjugates 1 BeadsConjugates 1 S-PE Sample (step 1) (step 1) (step 2) (step 3) SDC PVC

Materials

(i) Analysis System

The BioPlex 200® analyzer (Bio-Rad, Marnes-la-Coquette, France) is usedaccording to the manufacturer's instructions. This immunoanalysismachine contains a flow cytometer and a Luminex 100™ detector (LuminexCorp., Austin, Tex., United States) and uses heterogeneous sets ofsuperparamagnetic particles. Each homogenous group of particles,polystyrene compounds and methacrylic acid (COOH function), and having asize of 8 μm in diameter, is manufactured with different percentages offluorochromes (CL1 and CL2) producing a unique identification codeassigned to each group of particles and detectable by the laser of theLuminex 100™ detector (Luminex Corp., Austin, Tex., United States).After the immunological reaction, the beads of one set pass one by onethrough a flow cell, at the center of a liquid sheath, to besimultaneously excited and read by two separate lasers. The measurementsare done upon the passage of each bead.

The laser with a 638 nm red ray excites the identification fluorochromes(CL1 and CL2) encrusted in the surface of each particle and thecomposite signal is interpreted to identify the analyte detected by theparticle. This laser therefore serves, by identifying the particlecategory, to identify the test in progress.

The laser with a 532 nm green ray excites the S-PE (streptavidin coupledwith Phycoerythrin) reporter and the emitted fluorescence isproportional to the quantity of reporter fixed on the particle. Thislaser therefore serves to measure the reactivity of the analyteimmobilized on said particle.

The software of the system converts the signal related to the presenceof the detection ligand into a relative fluorescence intensity (RFI)value. A ratio may be calculated in order to rate the resultqualitatively, as positive or negative.

(ii) Solid Phase (Beads)

A set of beads including six separate groups of Luminex™superparamagnetic particles (Luminex Corp., Austin, Tex., United States)is used. This set of beads comprises a group of beads for controllingthe deposition of a sample (SDC beads), a group of beads for controllingthe deposition of a detection ligand of an analyte (PVC beads) and 4groups of beads for detecting an analyte (analytes to be assayed: A1,A2, A3 and A4).

Each group of particles is coated with a specific capture ligand of aparticular test. Each capture ligand is coupled using ahetero-bifunctional reagent.

The SDC beads are covered with a soluble anti-receptor mouse monoclonalantibody of the Transferrin (Fitzgerald, United States) immobilized at 1μg/mg of particles.

The PVC beads are covered with an anti-Digoxigenin sheep polyclonalantibody (Abcam, United States) immobilized at 5 μg/mg of particles.

The detection beads of the analytes A1, A2, A3 and A4 are covered withone or several specific capture ligands of the analytes to be detected.

(iii) Detection Ligands

The detection ligand of the control compound (the SDC control),pAb-anti-Tf-biot, is an anti-Transferrin sheep polyclonal antibody(Bio-Rad, Barnes la Coquette, France) coupled to biotin (ThermoScientific, France) using a hetero-bifunctional reagent known in itselfby those skilled in the art.

The detection ligand of the additive (of the PVC control),pAb-anti-DIG-biot, is an anti-Digoxigenin sheep polyclonal antibody(Abcam, France) coupled to biotin (Pierce, United States) using ahetero-bifunctional reagent known in itself by those skilled in the art.

(iv) Additive

The BSA-DIG additive is Digoxigenin (Sigma, France) grafted on a carriermolecule, in this example Bovine Serum Albumin (Millipore, France),using a hetero-bifunctional reagent known in itself by those skilled inthe art.

(v) Reporter

The S-PE reporter is streptavidin (Roche, Germany) coupled to thePhycoerythrin (Cyanotech, Hawaii, United States), using ahetero-bifunctional reagent known in itself by those skilled in the art.

(vi) Diluents

vi.1. Diluent of the Superparamagnetic Particles

Tris buffer solution 50 mM, pH 7.5, containing: NaCl 150 mM, EDTA 20 mM,Bovine Serum Albumin at 10%, mouse IgG (Meridian, United States) at 500μg/mL, Octyl-n-Glucoside at 0.10%, NaN3 at 0.095%.

vi.2. Diluent of Conjugates 1

Tris buffer solution 50 mM, pH 7.5, containing: NaCl 150 mM; EDTA 20 mM,Chaps 0.1%, Glycerol 10%, NaN3 at 0.095%.

vi.3. Diluent of Conjugates 2

Citrate buffer solution 50 mM, pH 6.7, containing: NaCl 150 mM, EDTA 5.6mM, Bovine Serum Albumin at 1%, Triton at 2%, Sheep serum at 10%, mouseIgG (Meridian, United States) at 500 μg/mL, Proclin 300™ (trademark ofthe company Supelco) at 0.5%, cow's milk (100% skim) at 15%, Glycerol10%, NaN3 at 0.095%.

vi.4. Diluent of the S-PE Reporter

Phosphate buffer, pH 7.4 containing: NaCl 150 mM, Tween 20™ (trademarkof the company Sigma) at 0.1%, Proclin 300™ (trademark of the companySupelco) at 0.5%, PEG 6000 2.75%, Bovine Serum Albumin 1%, Normal SheepSerum 1%, NaN3 at 0.095%.

vi.5. Washing Solution

Tris 10 mM buffer solution, pH 7.4, containing: NaCl 218 mM, Tween 20™(trademark of the company Sigma) at 0.1%, Proclin 300™ (trademark of thecompany Supelco) at 0.002%.

(vii) Reaction Dishes

The immunological reactions take place in the wells of 96-wellmicroplates made from poly propylene having a maximum volume of 355 μLper well.

(viii) Samples

The negative samples (serum or plasma) used come from the French bloodagency in Lille.

Methods

The multiplex format test protocol comprises assaying 4 differentanalytes, the assay of analytes A1 and A2 being done in oneimmunological time, the assay of analytes A3 and A4 being done in twoimmunological times.

The format of the tests is shown in FIGS. 2 and 4, in which S-PE is usedin place of S-POD.

Step 1:

1. In each well of a microplate are successively distributed:

+100 μL of sample

+25 μl of diluent of the conjugates 1 containing:

-   -   +/− BSA-DIG    -   +/− the detection ligands of analytes A1 and A2

+25 μL of immunoreactive superparamagnetic particles (mixture ofparticles, 1.2 μg per type of beads: SDC, PVC+/− beads of the analytesto be assayed)

2. The mixture is incubated for 40 minutes at 37° C. with agitation.

3. The following washing steps are carried out: separation of the solidand liquid phases by magnetization and 3 successive washes with at least300 μl of wash solution. In the last wash, the particles are put back insuspension.

Step 2:

4. Distributed in each reaction well is 90 μl of diluent of conjugates 2containing:

+/− the detection ligand of the pAb-anti-Tf-biot control compound

+/− the detection ligand of the pAb-anti-DIG-biot additive

+/− the detection ligands of analytes A3 and A4

5. The mixture is incubated for 15 minutes at 37° C. with agitation.

6. The wash steps (idem point 3) are carried out.

Step 3:

7. 90 μL of the S-PE reporter is distributed in each reaction well.

8. The mixture is incubated for 15 minutes at 37° C. with agitation.

9. The wash steps (idem point 3) are carried out.

10. The particles are put back in suspension in each reaction well whileadding 120 μL of washing solution, then the microplate is agitated.

11. The suspension of particles in each well is aspirated by the flowcytometer.

12. The suspension of particles in each well is read using two laserrays.

13. The results of the readings are processed directly by the flowcytometer and recorded in Relative Fluorescence Intensity (RFI) units.

14. To interpret the results, for each sample, a ratio is calculatedwith respect to a threshold value (“cutoff”).

Calculation of the Ratio

The SDC ratio of the samples is calculated as follows:

${{Sample}\mspace{14mu} {SDC}\mspace{14mu} {ratio}} = \frac{{RFI}\mspace{14mu} {signal}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {sample}}{{SDC}\mspace{14mu} {threshold}\mspace{14mu} {value}}$

Likewise, the PVC ratio of the samples is calculated as follows:

${{Sample}\mspace{14mu} {PVC}\mspace{14mu} {ratio}} = \frac{{RFI}\mspace{11mu} {signal}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {sample}}{{PVC}\mspace{14mu} {threshold}\mspace{14mu} {value}}$

The samples having ratios (SDC or PVC) above 1 are declared “valid”;those for which the ratios are below 1 are declared “not valid”.

The SDC and PVC threshold value has been established according to astatistical study described in the results below.

Results

(i) SDC System in Simplex Format

In the system, the detection beads of analytes A1 to A4 and theirdetection ligands are not used.

The study of 94 samples makes it possible to determine the thresholdvalue of the SDC system (cf. FIG. 7). The threshold value of the SDCsystem is calculated by subtracting 3 times the standard deviation ofthe signal of the sample population from the average value of the signalof the population.

TABLE 2 Statistics of the SDC signal for the population of 94 samplesand calculation of the threshold value Average (RFI) 334 Standarddeviation (σ) (RFI) 44.02 Variation coefficient (CV) in % 13.2% Maximumof the population (RFI) 427 Minimum of the population (RFI) 239Threshold value = Average − 3 σ 201.5 (RFI)

The response of the SDC system is measured in the case of a nominalmethod (Deposited volume of sample=100 μL, Conjugates 2 volume=90 μL,S-PE volume=90 μL) (case no. 1 of table 3). Cases 2, 3, 4 of table 3 aredowngraded methods: case 2=absence of sample deposition, case 3=absenceof deposition of conjugates 2, case 4=absence of S-PE deposition.

In this case, the mixture comprising the “conjugates 2” comprises 90 μLof diluent of the conjugates 2 containing the detection ligand of thepAb-anti-Tf-biot control compound.

TABLE 3 Summary of the SDC ratios obtained during a nominal method(case 1) and downgraded methods (cases 2, 3 and 4) Sample ConjugatesVolume Status Volume 2 Volume S-PE SDC VALID/NOT Case (μL) (μL) (μL)RFIs* ratio VALID 1 100 90 90 331 1.6 VALID 2 0 90 90 31 0.2 NOT VALID 3100 0 90 29 0.1 NOT VALID 4 100 90 0 29 0.1 NOT VALID *RFIs: RelativeFluorescence Intensities

Cases 2, 3, 4 lead to SDC ratios lower than 1, which makes it possibleto invalidate the measurements from these downgraded methods.

(ii) Impact of the SDC System on Performance in Multiplex Format

In the SDC system in multiplex format (MPX), the detection beads ofanalytes A1 to A4 and the corresponding detection ligands are used.

By comparing the RFI signals obtained between a MPX format without SDCversus MPX with SDC (cf. table 4), the addition of a SDC format does notaffect the performance of a multiplex including 4 analytes (i.e., thedeviations in % between the RFI signals of a MPX format without SDCversus MPX with SDC are comprised in an interval+/−20%, which isconsidered statistically acceptable).

TABLE 4 Comparison in % of the RFI signals obtained between a MPX formatwithout SDC versus a MPX format with SDC Number of samples used for thecalculations Analyte 1 Analyte 2 Analyte 3 Analyte 4 Standard 4 positivesamples −5.2% deviation in % Analyte 1 between the 3 positive samples−3.0% RFI signal Analyte 2 measured in 5 positive samples −2.2% MPXwithout Analyte 3 SDC versus 2 positive samples −19.9% MPX with Analyte4 SDC 32 negative samples 0.1% 1.3% −0.1% −2.5% for the 4 Analytes

(iii) PVC in Simplex Format

As before, in this format, the detection beads of analytes A1 to A4 andthe corresponding detection ligands are not used.

The study of 38 samples makes it possible to determine the thresholdvalue of the PVC system (cf. FIG. 8).

The threshold value of the PVC system is calculated by subtracting 3times the standard deviation of the signal of the sample population fromthe average value of the signal of the population (cf. table 5).

TABLE 5 Statistics of the PVC signal for the population of 38 samplesand calculation of the threshold value Average (RFI) 356 Standarddeviation (σ) (RFI) 70.92 Variation coefficient (CV) in % 19.9% Maximumof the population (RFI) 510 Minimum of the population (RFI) 222Threshold value = Average − 3 σ 143.4 (RFI)

The response of the PVC system is measured in the case of a nominalmethod (Deposited volume of sample=100 μL, Conjugates 1 volume=90 μL,S-PE volume=90 μL) (case no. 1 of table 6). Cases 2, 3 of table 6 aredowngraded methods: case 2=absence of conjugates 1 deposition, case3=absence of S-PE deposition.

TABLE 6 Summary of the PVC ratios obtained during a nominal method(case 1) and downgraded methods (cases 2, 3) Volume Conjugates 1 S-PEStatus Case Volume (μL) (μL) RFIs* Ratio VALID/NOT VALID 1 90 90 356 2.5VALID 2 0 90 93 0.6 NOT VALID 3 90 0 30 0.2 NOT VALID

Cases 2 and 3 lead to PVC ratios lower than 1, which makes it possibleto invalidate the measurements from these downgraded methods.

(iv) Impact of the Simplex Versus Multiplex Mode on the Performance ofthe PVC System

In the PVC system in multiplex format, the detection beads of analytesA1 to A4 and the corresponding detection ligands are used.

The performance of the PVC system is similar in simplex mode andmultiplex mode (cf. table 7).

TABLE 7 Comparison of the PVC performance in simplex versus multiplexmode 4 Analytes, PVC (multiplex) PVC (simplex) Average RFI of 38 samples(RFI) 356 320 CV (%) 19.9% 20.6% Max (RFI) 510 472 Min (RFI) 222 194Threshold value = Average − 3 σ 143.4 121.7 (RFI)

Furthermore, by comparing the RFI signals obtained between a MPX formatwithout SDC or PVC versus a MPX format with SDC and PVC, it appears thatthe addition of SDC and PVC tests does not affect the performance of amultiplex including 4 analytes to be assayed (i.e., the deviations in %between the RFI signals of a MPX format without SDC or PVC versus a MPXformat with SDC and PVC are comprised in an interval+/−20%, which isconsidered statistically acceptable).

Example 2 Spotting Method

Materials and Methods

The three controls described in this example make it possible tovalidate (cf. table 8):

-   -   for the SDC (“Sample Deposit Control” or “control of the        deposition of a sample”): the deposition of the sample and also        step 2 (deposition of the conjugates 2), step 3 (deposition of        the S-POD reporter) and step 4 (deposition of the Luminol        substrate).    -   for the PVC (“Process Verification Control” or “control of the        deposition of a detection ligand of an analyte”): the        depositions of step 1 (deposition of the additive and deposition        of the conjugates 1), step 3 (deposition of the S-POD reporter)        and step 4 (deposition of the Luminol substrate), and    -   for the RVC (“Revelation Verification Control” or “control of        the deposition of a reporter”): the revelation step, by        controlling both step 3 (deposition of the S-POD reporter) and        step 4 (deposition of the Luminol substrate).

TABLE 8 Steps controlled by the SDC, PVC and RVC controls Conju- Sam-Additive conjugates 1 gates 2 S-POD Luminol ple (step 1) (step 1) (step2) (step 3) (step 4) SDC PVC RVC

Materials

(i) Analysis System

The technology used for this system is an innovative nanospotting onbiochip multiplex technology (cf. definition below), with developing bychemiluminescence owing to a reporter marked by the enzyme of thehorseradish peroxidase and developed by a substrate of the luminol type.

The term “biochip” is a collection of miniaturized test sites (or“micro-array”) arranged on a solid support that makes it possible toperform many tests at the same time in order to obtain a faster rhythm.

Within each well of a microplate (Greiner, Germany), a spotter robot isused to deposit 50 nL drops of a protein solution containing proteins orantibodies specific to the analyte to be assayed (A1, A2, A3, A4, A5,SDC, RVC and PVC) (cf. FIG. 6). The bottom of each well of thesemicroplates has protein and peptide adsorption capacities known inthemselves by those skilled in the art. The spots thus obtained aresaturated with a saturation solution known in itself by those skilled inthe art.

It is next possible to perform a traditional immunological reaction in 1or 2 immunological times within these biochips.

After the immunological reaction, the addition of the developingsubstrate causes a light emission. Indeed, the oxidation of the luminolby enzymatic catalysis leads to a light emission proportional to thequantity of Streptavidin-peroxidase reporter fixed by the spot. Theacquisition of the signal is done by a scientific camera. The resultingimage is then analyzed in order to determine the intensity of theluminescence produced by each geographical zone of the bottom of thewell corresponding to each spot (addressing information).

The software of the system converts the signal measured by spot into avalue in Relative Luminescence Units (RLU). A ratio may be calculated inorder to rate the result qualitatively, as positive or negative, asoutlined below.

(ii) Solid Phase (Spots)

The different control spots are:

-   -   the SDC spot, comprising a soluble anti-receptor mouse        monoclonal antibody of the Transferrin (Fitzgerald, United        States) immobilized at 50 μg/mL,    -   the PVC spot, comprising a soluble anti-Digoxigenin mouse        monoclonal antibody (Covalab, France) immobilized at 25 μg/mL,        and    -   the RVC spot comprising an anti-KLH (Keyhole Limpet Hemocyanin)        mouse monoclonal antibody (Genway, United States) coupled to        biotin (Thermo Scientific, France) using a hetero-bifunctional        reagent known in itself by those skilled in the art and        immobilized at 1 μg/mL.

(iii) Detection Ligands

The detection ligand of the control compound (relative to the SDCcontrol), pAb-anti-Tf-biot, is an anti-Transferrin sheep polyclonalantibody (Bio-Rad, Barnes la Coquette, France) coupled to biotin (ThermoScientific, France) using a hetero-bifunctional reagent known in itselfby those skilled in the art.

The detection ligand of the additive (relative to the PVC control),mAb-anti-DIG-biot, is an anti-Digoxigenin mouse monoclonal antibodycoupled to biotin (Covalab, France).

(iv) Additive

The BSA-DIG additive is Digoxigenin (Sigma, France) grafted on a carriermolecule, in this example Bovine Serum Albumin (Millipore, France). Thecoupling is done using a hetero-bifunctional reagent known in itself bythose skilled in the art.

(v) Reporter

The S-POD reporter is streptavidin (Roche, Germany) coupled withPeroxidase (Roche Germany) according to the method described by P.Nakane and A. Kawaoi [J Histochem Cytochem (1974) Vol. 22, No. 12. pp.1084-1091), known in itself by those skilled in the art.

(vi) Diluents

a) Diluent of the Additive

Tris buffer solution 50 mM, pH 7.5, containing: NaCl 150 mM, EDTA 20 mM,mouse IgG (Meridian, United States) at 500 μg/mL, Cow's milk (100% skim)at 15%, Sheep serum at 10%, NaN3 at 0.095%.

b) Diluent of conjugates 1

Tris buffer solution 50 mM, pH 7.5, containing: NaCl 150 mM; EDTA 20 mM,Chaps 0.1%, Glycerol 10%, NaN3 at 0.095%.

c) Diluent of conjugates 2

Citrate buffer solution 50 mM, pH 6.7, containing: NaCl 150 mM, EDTA 5.6mM, Triton at 2%, Sheep serum at 10%, mouse IgG 500 μg/mL, Proclin 300™(trademark of the company Supelco) at 0.5%, cow's milk (100% skim) at15%, Glycerol 10%. NaN3 at 0.095%.

d) Diluent of the S-POD Reporter

Citrate buffer solution 50 mM, pH 6.7, containing: NaCl 2053 mM, Tween20™ (trademark of the company Sigma) at 0.5%, Proclin 300™ (trademark ofthe company Supelco) at 0.5%, cow's milk (100% skim) at 7%, Glycerol20%.

e) Wash Solution

Tris 10 mM buffer solution, pH 7.4, containing: NaCl 218 mM, Tween 20™(trademark of the company Sigma) at 0.1%, Proclin 300™ (trademark of thecompany Supelco) at 0.002%.

f) Developing Substrate

The ELISTAR ETA C Ultra ELISA developing substrate (Cyanagen, Italy) ismade up of two solutions: XLSE024L Luminol enhancer solution (A) andXLSE024P Peroxide solution (B).

(vii) Reaction Dishes

The immunological reactions take place in 96-well microplates made frompolystyrene having a maximum volume of 392 μL per well.

(viii) Samples

The negative samples (serum or plasma) used come from the French bloodagency in Lille.

Methods

The test protocol comprises the following steps.

Step 1:

1. In each well of a microplate (comprising the spots) are successivelydistributed:+20 μl of diluent of the additive containing the BSA-DIG additive+20 μl of diluent of the conjugates 1 comprising:

+ the detection ligand of the mAb-anti-DIG-biot additive, and

+ the detection ligands of analytes 1, 2 and 3 to be assayed

+40 μl of sample2. The mixture is incubated for 40 minutes at 37° C. with agitation.3. Three successive washes with at least 400 μl of wash solution aredone.

Step 2:

4. Distributed in each reaction well is 50 μl of diluent of conjugates 2containing:

+ the detection ligand of the pAb-anti-Tf-biot control compound

+ the detection ligands of analytes 4 and 5 to be assayed.

5. The mixture is incubated for 15 minutes at 37° C. with agitation.6. The wash steps (idem point 3) are carried out.

Step 3:

7. 50 μL of the S-POD reporter is distributed in each reaction well.8. The mixture is incubated for 15 minutes at 37° C. with agitation.

Step 4:

9. 25 μL of developing solution “B” is distributed in each reactionwell.10. 25 μL of developing solution “A” is distributed in each reactionwell.10. The mixture is incubated for 1 minute at 37° C. with agitation.11. The acquisition of the luminescence signal is done for 180 seconds.12. The results of the readings are processed directly by an imageanalysis system and recorded in Relative Light Units (RLU).13. To interpret the results, for each sample, a ratio is calculatedwith respect to a threshold value (or “cutoff”).

Calculation of the Ratio

Two Analysis Modes are Illustrated Below:

Analysis Mode 1: A Single Threshold Value

The SDC ratio of the samples is calculated as follows:

${{Sample}\mspace{14mu} {SDC}\mspace{14mu} {ratio}} = \frac{{RLU}\mspace{14mu} {signal}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {sample}}{{SDC}\mspace{14mu} {threshold}\mspace{14mu} {value}}$

Likewise, the PVC ratio of the samples is calculated:

${{Sample}\mspace{14mu} {PVC}\mspace{14mu} {ratio}} = \frac{{RLU}\mspace{14mu} {signal}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {sample}}{{PVC}\mspace{14mu} {threshold}\mspace{14mu} {value}}$

Similarly, the RVC ratio of the samples is calculated:

${{Sample}\mspace{14mu} {RVC}\mspace{14mu} {ratio}} = \frac{{RLU}\mspace{14mu} {signal}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {sample}}{{RVC}\mspace{14mu} {threshold}\mspace{14mu} {value}}$

The samples having ratios (SDC or PVC or RVC) above 1 are declared“valid”; those for which the ratios are below 1 are declared “notvalid”. The SDC, PVC and RVC threshold value has been establishedaccording to a statistical study described in the results chapter below.

Analysis Mode 2: Two Threshold Values

Two SDC ratios of the samples are calculated as follows:

${{Sample}\mspace{14mu} {SDC}\mspace{14mu} {ratio}\mspace{14mu} \left( {{threshold} -} \right)} = \frac{{RLU}\mspace{14mu} {signal}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {sample}}{{SDC}\mspace{14mu} \left( {{threshold} -} \right)\mspace{14mu} {value}}$${{Sample}\mspace{14mu} {SDC}\mspace{14mu} {ratio}\mspace{14mu} \left( {{threshold} +} \right)} = \frac{{RLU}\mspace{14mu} {signal}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {sample}}{{SDC}\mspace{14mu} \left( {{threshold} +} \right)\mspace{14mu} {value}}$

Likewise, the PVC ratios of the samples are calculated:

${{Sample}\mspace{14mu} {PVC}\mspace{14mu} {ratio}\mspace{14mu} \left( {{threshold} -} \right)} = \frac{{RLU}\mspace{14mu} {signal}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {sample}}{{PVC}\mspace{14mu} \left( {{threshold} -} \right)\mspace{14mu} {value}}$${{Sample}\mspace{14mu} {PVC}\mspace{14mu} {ratio}\mspace{14mu} \left( {{threshold} +} \right)} = \frac{{RLU}\mspace{14mu} {signal}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {sample}}{{PVC}\mspace{14mu} \left( {{threshold} +} \right)\mspace{14mu} {value}}$

Similarly, the RVC ratios of the samples are calculated:

${{Sample}\mspace{14mu} {RVC}\mspace{14mu} {ratio}\mspace{14mu} \left( {{threshold} -} \right)} = \frac{{RLU}\mspace{14mu} {signal}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {sample}}{{RVC}\mspace{14mu} \left( {{threshold} -} \right)\mspace{14mu} {value}}$${{Sample}\mspace{14mu} {RVC}\mspace{14mu} {ratio}\mspace{14mu} \left( {{threshold} +} \right)} = \frac{{RLU}\mspace{14mu} {signal}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {sample}}{{RVC}\mspace{14mu} \left( {{threshold} +} \right)\mspace{14mu} {value}}$

The samples having ratios (threshold−) (SDC or PVC or RVC) above 1 aredeclared “valid”; those for which the ratios are below 1 are declared“not valid”.

The samples having ratios (threshold+) (SDC or PVC or RVC) below 1 aredeclared “valid”; those for which the ratios are above 1 are declared“not valid”.

The SDC, PVC and RVC threshold value has been established according to astatistical study described in the results chapter below.

Results

The multiplex described in this example includes 5 analytes to beassayed, 3 analytes whereof the detection ligands are added in step 1(A1, A2 and A3), 2 analytes whereof the detection ligands are added instep 2 (A4 and A5) and the three SDC, PVC and RVC controls.

Table 9 groups together the scenarios that may be encountered uponisolated or cumulative invalidation of the SDC, PVC and RVC controls.

The responses of the SDC, PVC and RVC controls are measured in the caseof a nominal method (Additive Volume=20 μL, Conjugates 1 Volume=20 μL,Deposited Sample Volume=40 μL, Conjugates 2 Volume=50 μL, S-PODVolume=50 μL, Luminol Volume=25 μL solution B+25 μL solution A) for 22samples.

The study of these 22 samples makes it possible to characterize the SPC,PVC, RVC response in terms of average spots, standard deviation, minimumand maximum value (cf. table 10a and 10b). The threshold values of theSDC, PVC and RVC controls are also calculated.

TABLE 9 Interpretation of all of the SDC, PVC and RVC controls andidentification of the deficient step in the method. SDC and SDC PVC andStatus PVC RVC RVC VALID/ Status Status Status NOT VALID/ VALID/VALID/NOT Deficient step at the source of the VALID NOT VALID NOT VALIDVALID invalidation of the method NOT VALID VALID NOT Sample depositionor deposition of VALID VALID detection ligands in step 2 VALID NOT VALIDNOT Additive deposition or deposition of VALID VALID detection ligandsin step 1 NOT NOT VALID NOT Sample deposition or deposition of VALIDVALID VALID detection ligands in step 2 and Additive deposition ordeposition of detection ligands in step 1 NOT NOT NOT NOT S-POD orLuminol deposition VALID VALID VALID VALID Furthermore, in thisscenario, the sample, additive, detection ligands of step 1, detectionligands of step 2 depositions cannot be validated or invalidated. VALIDVALID NOT Cases An not valid RVC signal involves an VALID unable toinvalidation of the SDC and PVC NOT VALID NOT arise VALID VALID VALIDNOT NOT VALID VALID

Analysis Mode 1: A Single Threshold Value

TABLE 10a Response of the SDC, PVC and RVC controls (average response,standard deviation, minimum and maximum value, threshold values) SDC PVCRVC Average response (RLUs) 998 2623 3200 Standard deviation (σ) (RLUs)124 154 214 Variation coefficient (CV) in % 12.4% 5.9% 6.7% Maximum ofthe population (RLUs) 1246 2997 3535 Minimum of the population (RLUs)798 2407 2773 Threshold value = Average − 3 σ 627 2162 2559 (RLUs)

Analysis Mode 2: Two Threshold Values

TABLE 10b Responses of the SDC, PVC and RVC controls (average response,standard deviation, minimum and maximum value, threshold values) SDC PVCRVC Average response (RLUs) 998 2623 3200 Standard deviation (σ) (RLUs)124 154 214 Variation coefficient (CV) in % 12.4% 5.9% 6.7% Maximum ofthe population (RLUs) 1246 2997 3535 Minimum of the population (RLUs)798 2407 2773 Value (threshold+) = Average − 3 σ 627 2162 2559 (RLUs)Value (threshold+) = Average + 3 σ 1368 3084 3841 (RLUs)

By combining the responses of the SDC, PVC, RVC controls and thecalculated threshold values (cf. table 10a and 10b), the SDC, PVC andRVC ratios are calculated for each sample (cf. table 11a and 11b).

Analysis Mode 1: A Single Threshold Value

TABLE 11a Responses of the SDC, PVC and RVC controls in RLUs and ratiosof a population of 22 samples. Status SDC PVC RVC SDC AND Status StatusStatus PVC AND SDC PVC RVC RVC Spec. SDC SDC VALID/NOT PVC PVC VALID/NOTRVC RVC VALID/NOT VALID/NOT No. RLUs ratio VALID RLUs Ratio VALID RLUsRatio VALID VALID 1 974 1.55 VALID 2997 1.39 VALID 3368 1.32 VALID VALID2 970 1.55 VALID 2407 1.11 VALID 3437 1.34 VALID VALID 3 863 1.38 VALID2489 1.15 VALID 2773 1.08 VALID VALID 4 1072 1.71 VALID 2724 1.26 VALID3216 1.26 VALID VALID 5 1246 1.99 VALID 2830 1.31 VALID 3340 1.31 VALIDVALID 6 1219 1.94 VALID 2617 1.21 VALID 3165 1.24 VALID VALID 7 10021.60 VALID 2743 1.27 VALID 3535 1.38 VALID VALID 8 1172 1.87 VALID 26701.24 VALID 3150 1.23 VALID VALID 9 835 1.33 VALID 2686 1.24 VALID 30821.20 VALID VALID 10 932 1.49 VALID 2575 1.19 VALID 2806 1.10 VALID VALID11 1061 1.69 VALID 2641 1.22 VALID 3274 1.28 VALID VALID 12 929 1.48VALID 2617 1.21 VALID 3200 1.25 VALID VALID 13 1098 1.75 VALID 2495 1.15VALID 3445 1.35 VALID VALID 14 996 1.59 VALID 2551 1.18 VALID 3382 1.32VALID VALID 15 876 1.40 VALID 2732 1.26 VALID 3459 1.35 VALID VALID 16896 1.43 VALID 2623 1.21 VALID 3310 1.29 VALID VALID 17 897 1.43 VALID2429 1.12 VALID 3219 1.26 VALID VALID 18 798 1.27 VALID 2611 1.21 VALID2977 1.16 VALID VALID 19 906 1.44 VALID 2440 1.13 VALID 2993 1.17 VALIDVALID 20 1007 1.61 VALID 2412 1.12 VALID 2894 1.13 VALID VALID 21 10931.74 VALID 2868 1.33 VALID 3294 1.29 VALID VALID 22 1108 1.77 VALID 25471.18 VALID 3083 1.20 VALID VALID

Analysis Mode 2: Two Threshold Values

TABLE 11b Responses of the SDC, PVC and RVC controls in RLUs and(threshold+) ratios, (threshold−) ratios of a population of 22 samples.SDC PVC RVC SDC SDC PVC PVC RVC RVC Spec. SDC ratio ratio PVC RatioRatio RVC Ratio Ratio No. RLUs (threshold−) (threshold+) RLUs(threshold−) (threshold+) RLUs (threshold−) (threshold+) 1 974 1.55 0.712997 1.39 0.97 3368 1.32 0.88 2 970 1.55 0.71 2407 1.11 0.78 3437 1.340.89 3 863 1.38 0.63 2489 1.15 0.81 2773 1.08 0.72 4 1072 1.71 0.78 27241.26 0.88 3216 1.26 0.84 5 1246 1.99 0.91 2830 1.31 0.92 3340 1.31 0.876 1219 1.94 0.89 2617 1.21 0.85 3165 1.24 0.82 7 1002 1.60 0.73 27431.27 0.89 3535 1.38 0.92 8 1172 1.87 0.86 2670 1.24 0.87 3150 1.23 0.829 835 1.33 0.61 2686 1.24 0.87 3082 1.20 0.80 10 932 1.49 0.68 2575 1.190.83 2806 1.10 0.73 11 1061 1.69 0.78 2641 1.22 0.86 3274 1.28 0.85 12929 1.48 0.68 2617 1.21 0.85 3200 1.25 0.83 13 1098 1.75 0.80 2495 1.150.81 3445 1.35 0.90 14 996 1.59 0.73 2551 1.18 0.83 3382 1.32 0.88 15876 1.40 0.64 2732 1.26 0.89 3459 1.35 0.90 16 896 1.43 0.65 2623 1.210.85 3310 1.29 0.86 17 897 1.43 0.66 2429 1.12 0.79 3219 1.26 0.84 18798 1.27 0.58 2611 1.21 0.85 2977 1.16 0.78 19 906 1.44 0.66 2440 1.130.79 2993 1.17 0.78 20 1007 1.61 0.74 2412 1.12 0.78 2894 1.13 0.75 211093 1.74 0.80 2868 1.33 0.93 3294 1.29 0.86 22 1108 1.77 0.81 2547 1.180.83 3083 1.20 0.80

The status of the interpretations from the (threshold+) and (threshold−)ratios of the SDC; PVC, RVC controls is “Valid” for all 22 samples. Thestatus of the interpretations from the SDC, PVC, RVC controls is “Valid”for all 22 samples.

A case study of methods done in downgraded mode is illustrated in tables12 and 13a and 13b. Six cases are presented:

-   -   Case 1=absence of sample deposition.    -   Case 2=absence of additive deposition.    -   Case 3=absence of conjugates 1 deposition.    -   Case 4=absence of conjugates 2 deposition.    -   Case 5=absence of S-POD deposition.    -   Case 6=absence of Luminol deposition.

Analysis Mode 1: A Single Threshold Value

Cases 1 and 4 lead to SDC ratios lower than 1, which makes it possibleto invalidate the measurements from these downgraded methods.

Cases 2 and 3 lead to PVC ratios lower than 1, which makes it possibleto invalidate the measurements from these downgraded methods.

Cases 5 and 6 lead to SDC, PVC and RVC ratios lower than 1, which makesit possible to invalidate the measurements from these downgradedmethods. The RVC signal makes it possible to specifically invalidate thedeveloping step (S-POD deposition and Luminol deposition). However, theinvalidation of the procedure by a RVC ratio lower than 1 implies anabsence of the systematic signal of the SDC and PVC controls (cf. grayzone of table 9). In this case, it is not possible to determine whetherthe depositions of the sample and the conjugates 2 (depositionscontrolled by SDC) or the depositions of additive and the conjugates 1(depositions controlled by PVC) have taken place correctly.

Analysis Mode 2: Two Threshold Values

Cases 1 and 4 lead to SVC (threshold−) ratios lower than 1, which makesit possible to invalidate the measurements from these downgradedmethods. In addition to case 1, the PVC (threshold+) ratio is greaterthan 1, which reflects an indirect impact of the absence of sample onthe PVC control.

Cases 2 and 3 lead to PVC (threshold−) ratios lower than 1, which makesit possible to invalidate the measurements from these downgradedmethods.

Cases 5 and 6 lead to SDC, PVC and RVC (threshold−) ratios lower than 1,which makes it possible to invalidate the measurements from thesedowngraded methods. The RVC signal makes it possible to specificallyinvalidate the developing step (S-POD deposition and Luminoldeposition). However, the invalidation of the procedure by a RVC(threshold−) ratio lower than 1 implies an absence of the systematicsignal of the SDC and PVC controls (cf. gray zone of table 9). In thiscase, it is not possible to determine whether depositions of additiveand the conjugates 1 (depositions controlled by PVC) have taken placecorrectly.

TABLE 12 Detail of the 6 downgraded method cases Step 1 Addi- Conju-Step 2 Step 3 Step 4 Sample tive gates Conjugates S-POD Volume VolumeVolume 1 Volume 2 Volume Volume Luminol Case (μL) (μL) (μL) (μL) (μL)(μL) 1 0 20 20 50 50 50 2 40 0 20 50 50 50 3 40 20 0 50 50 50 4 40 20 200 50 50 5 40 20 20 50 0 50 6 40 20 20 50 50 0

Analysis Mode 1: A Single Threshold Value

TABLE 13a Responses of the SDC, PVC and RVC controls in RLU and ratio ofa population of 22 samples SDC AND SDC PVC RVC PVC AND SDC PVC RVC RVCVALID/ VALID/ VALID/ VALID/ NOT NOT NOT NOT SDC SDC VALID PVC PVC VALIDRVC RVC VALID VALID Case RLUs ratio (NV) RLUs Ratio (NV) RLUs Ratio (NV)(NV) 1 37 0.06 NV 3939 1.82 VALID 3458 1.35 VALID NV 2 991 1.58 VALID 720.03 NV 3569 1.39 VALID NV 3 983 1.57 VALID 59 0.03 NV 3046 1.19 VALIDNV 4 18 0.03 NV 2727 1.26 VALID 3655 1.43 VALID NV 5 5 0.01 NV 4 0.00 NV5 0.00 NV NV 6 6 0.01 NV 7 0.00 NV 5 0.00 NV NV

Analysis Mode 2: Two Threshold Values

TABLE 13b Responses of the SDC, PVC and RVC controls in RLU and ratio ofa population of 22 samples SDC PVC RVC SDC PVC RVC SDC SDC VALID/ PVCPVC VALID/ RVC RVC VALID/ SDC AND PVC ratio ratio NOT Ratio Ratio NOTRatio Ratio NOT AND RVC SDC (thresh- (thresh- VALID PVC (thresh-(thresh- VALID RVC (thresh- (thresh- VALID VALID/NOT Case RLUs old−)old+) (NV) RLUs old−) old+) (NV) RLUs old−) old+) (NV) VALID (NV) 1 370.06 0.03 NV 3939 1.82 1.28 NV 3458 1.35 0.90 VALID NV (directmeasurement), NV (indirect measurement of the effect of the absence ofsample on PVC) 2 991 1.58 0.72 VALID 72 0.03 0.02 NV 3569 1.39 0.93VALID NV 3 983 1.57 0.72 VALID 59 0.03 0.02 NV 3046 1.19 0.79 VALID NV 418 0.03 0.01 NV 2727 1.26 0.88 VALID 3655 1.43 0.95 VALID NV 5 5 0.010.00 NV 4 0.00 0.00 NV 5 0.00 0.00 NV NV 6 6 0.01 0.00 NV 7 0.00 0.00 NV5 0.00 0.00 NV NV

Furthermore, by comparing the RLU signals obtained between a MPX formatwithout SDC or PVC versus a MPX format with SDC and PVC, it appears thatthe addition of SDC and PVC tests does not affect the performance of amultiplex including 4 analytes to be assayed (i.e., the deviations in %between the RLU signals of a MPX format without SDC or PVC versus a MPXformat with SDC and PVC are comprised in an interval+/−20%, which isconsidered statistically acceptable).

1-13. (canceled)
 14. A solid support appropriate for a multiplexanalysis of at least one sample, said solid support comprising at leastone compartment, said compartment comprising at least one control spotand at least two detection spots for an analyte, characterized in thatsaid control spot is selected from the group consisting of a spot forcontrolling the deposition of a sample, a spot for controlling thedeposition of a detection ligand of an analyte and a spot forcontrolling the deposition of a reporter.